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

PubMed

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

2014-06-27

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

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

PubMed

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

2003-03-01

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

Serum- and Glucocorticoid-Inducible Kinase 1 Confers Protection in Cell-Based and in In Vivo Neurotoxin Models via the c-Jun N-Terminal Kinase Signaling Pathway

PubMed Central

Iqbal, Sarah; Howard, Shannon

2015-01-01

Serum glucocorticoid kinase 1 (SGK1) has been shown to be protective in models of Parkinson's disease, but the details by which it confers benefit is unknown. The current study was designed to investigate the details by which SGK1 confers neuroprotection. To do this we employed a cellular neurodegeneration model to investigate c-Jun N-terminal kinase (JNK) signaling and endoplasmic reticulum (ER) stress induced by 6-hydroxydopamine. SGK1-expressing adenovirus was created and used to overexpress SGK1 in SH-SY5Y cells, and dexamethasone was used to increase endogenous expression of SGK1. Oxidative stress, mitochondrial dysfunction, and cell death were monitored to test the protective effect of SGK1. To investigate the effect of SGK1 overexpression in vivo, SGK1-expressing adenovirus was injected into the striatum of mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and protection of dopaminergic neurons was quantitatively assessed by tyrosine hydroxylase immunohistochemistry. SGK1 overexpression was found to decrease reactive oxygen species generation, alleviate mitochondrial dysfunction, and rescue cell death in vitro and in vivo by inactivating mitogen-activated protein kinase kinase 4 (MKK4), JNK, and glycogen synthase kinase 3β (GSK3β) and thereby decreasing ER and oxidative stress. These results suggest that therapeutic strategies for activation of SGK1 may have the potential to be neuroprotective by deactivating the JNK and GSK3β pathways. PMID:25825522

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.

PubMed

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

Vincristine activates c-Jun N-terminal kinase in chronic lymphocytic leukaemia in vivo

PubMed Central

Bates, Darcy J P; Lewis, Lionel D; Eastman, Alan; Danilov, Alexey V

2015-01-01

Aims The authors’ aim was to conduct a proof-of-principle study to test whether c-Jun N-terminal kinase (JNK) phosphorylation and Noxa induction occur in peripheral blood chronic lymphocytic leukaemia (CLL) cells in patients receiving a vincristine infusion. Methods Patients with CLL received 2 mg vincristine by a 5-min intravenous infusion. Blood samples were collected at baseline and up to 6 h after the vincristine infusion, and assayed for JNK activation, Noxa induction and vincristine plasma concentrations. Results Ex vivo treated peripheral CLL cells activated JNK in response to 10–100 nM vincristine in 6 h. Noxa protein expression, while variable, was also observed over this time frame. In CLL patients, vincristine infusion led to rapid (<1 h) JNK phosphorylation in peripheral blood CLL cells which was sustained for at least 4–6 h after the vincristine infusion. Noxa protein expression was not observed in response to vincristine infusion. Conclusions This study confirmed that vincristine can activate JNK but not induce Noxa in CLL cells in vivo. The results suggest that novel JNK-dependent drug combinations with vincristine warrant further investigation. PMID:25753324

c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis

DTIC Science & Technology

2015-03-01

1 AWARD NUMBER: W81XWH-12-1-0431 TITLE: “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis ” PRINCIPAL...TITLE AND SUBTITLE “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Scelerosis” 5a. CONTRACT NUMBER 5b. GRANT NUMBER... Lateral   Sclerosis ”   Final  Report:  Project  Period  Sept  2012-­‐Dec  2014     Personnel  List:     Feng,  Yangbo

C-Jun N-terminal kinase signalling pathway in response to cisplatin.

PubMed

Yan, Dong; An, GuangYu; Kuo, Macus Tien

2016-11-01

Cisplatin (cis diamminedichloroplatinum II, cDDP) is one of the most effective cancer chemotherapeutic agents and is used in the treatment of many types of human malignancies. However, inherent tumour resistance is a major barrier to effective cisplatin therapy. So far, the mechanism of cDDP resistance has not been well defined. In general, cisplatin is considered to be a cytotoxic drug, for damaging DNA and inhibiting DNA synthesis, resulting in apoptosis via the mitochondrial death pathway or plasma membrane disruption. cDDP-induced DNA damage triggers signalling pathways that will eventually decide between cell life and death. As a member of the mitogen-activated protein kinases family, c-Jun N-terminal kinase (JNK) is a signalling pathway in response to extracellular stimuli, especially drug treatment, to modify the activity of numerous proteins locating in the mitochondria or the nucleus. Recent studies suggest that JNK signalling pathway plays a major role in deciding the fate of the cell and inducing resistance to cDDP-induced apoptosis in human tumours. c-Jun N-terminal kinase regulates several important cellular functions including cell proliferation, differentiation, survival and apoptosis while activating and inhibiting substrates for phosphorylation transcription factors (c-Jun, ATF2: Activating transcription factor 2, p53 and so on), which subsequently induce pro-apoptosis and pro-survival factors expression. Therefore, it is suggested that JNK signal pathway is a double-edged sword in cDDP treatment, simultaneously being a significant pro-apoptosis factor but also being associated with increased resistance to cisplatin-based chemotherapy. This review focuses on current knowledge concerning the role of JNK in cell response to cDDP, as well as their role in cisplatin resistance. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

The contribution of c-Jun N-terminal kinase activation and subsequent Bcl-2 phosphorylation to apoptosis induction in human B-cells is dependent on the mode of action of specific stresses

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

Muscarella, Donna E.; Bloom, Stephen E.

2008-04-01

The c-Jun N-terminal kinase (JNK) pathway can play paradoxical roles as either a pro-survival or a pro-cell death pathway depending on type of stress and cell type. The goal of the present study was to determine the role of JNK pathway signaling for regulating B-cell apoptosis in two important but contrasting situations-global proteotoxic damage, induced by arsenite and hyperthermia, versus specific microtubule inhibition, induced by the anti-cancer drug vincristine, using the EW36 B-cell line. This cell line over-expresses the Bcl-2 protein and is a useful model to identify treatments that can overcome multi-drug resistance in lymphoid cells. Exposure of EW36more » B-cells to arsenite or lethal hyperthermia resulted in activation of the JNK pathway and induction of apoptosis. However, pharmacological inhibition of the JNK pathway did not inhibit apoptosis, indicating that JNK pathway activation is not required for apoptosis induction by these treatments. In contrast, vincristine treatment of EW36 B-cells resulted in JNK activation and apoptosis that was suppressed by JNK inhibition. A critical difference between the two types of stress treatments was that only vincristine-induced JNK activation resulted in phosphorylation of Bcl-2 at threonine-56, a modification that can block its anti-apoptotic function. Importantly, Bcl-2 phosphorylation was attenuated by JNK inhibition implicating JNK as the upstream kinase. Furthermore, arsenite and hyperthermia treatments activated a p53/p21 pathway associated with apoptosis induction, whereas vincristine did not activate this pathway. These results reveal two stress-activated pathways, one JNK-dependent and another JNK-independent, either of which can bypass Bcl-2 mediated resistance, resulting in cell death.« less

Mu opioid receptor stimulation activates c-Jun N-terminal kinase 2 by distinct arrestin-dependent and independent mechanisms.

PubMed

Kuhar, Jamie Rose; Bedini, Andrea; Melief, Erica J; Chiu, Yen-Chen; Striegel, Heather N; Chavkin, Charles

2015-09-01

G protein-coupled receptor desensitization is typically mediated by receptor phosphorylation by G protein-coupled receptor kinase (GRK) and subsequent arrestin binding; morphine, however, was previously found to activate a c-Jun N-terminal kinase (JNK)-dependent, GRK/arrestin-independent pathway to produce mu opioid receptor (MOR) inactivation in spinally-mediated, acute anti-nociceptive responses [Melief et al.] [1]. In the current study, we determined that JNK2 was also required for centrally-mediated analgesic tolerance to morphine using the hotplate assay. We compared JNK activation by morphine and fentanyl in JNK1(-/-), JNK2(-/-), JNK3(-/-), and GRK3(-/-) mice and found that both compounds specifically activate JNK2 in vivo; however, fentanyl activation of JNK2 was GRK3-dependent, whereas morphine activation of JNK2 was GRK3-independent. In MOR-GFP expressing HEK293 cells, treatment with either arrestin siRNA, the Src family kinase inhibitor PP2, or the protein kinase C (PKC) inhibitor Gö6976 indicated that morphine activated JNK2 through an arrestin-independent Src- and PKC-dependent mechanism, whereas fentanyl activated JNK2 through a Src-GRK3/arrestin-2-dependent and PKC-independent mechanism. This study resolves distinct ligand-directed mechanisms of JNK activation by mu opioid agonists and understanding ligand-directed signaling at MOR may improve opioid therapeutics. Copyright © 2015 Elsevier Inc. All rights reserved.

Fenofibrate inhibits aldosterone-induced apoptosis in adult rat ventricular myocytes via stress-activated kinase-dependent mechanisms

PubMed Central

De Silva, Deepa S.; Wilson, Richard M.; Hutchinson, Christoph; Ip, Peter C.; Garcia, Anthony G.; Lancel, Steve; Ito, Masa; Pimentel, David R.; Sam, Flora

2009-01-01

Aldosterone induces extracellular signal-regulated kinase (ERK)-dependent cardiac remodeling. Fenofibrate improves cardiac remodeling in adult rat ventricular myocytes (ARVM) partly via inhibition of aldosterone-induced ERK1/2 phosphorylation and inhibition of matrix metalloproteinases. We sought to determine whether aldosterone caused apoptosis in cultured ARVM and whether fenofibrate ameliorated the apoptosis. Aldosterone (1 μM) induced apoptosis by increasing terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive nuclei in ARVM. Spironolactone (100 nM), an aldosterone receptor antagonist, but not RU-486, a glucocorticoid receptor, inhibited aldosterone-mediated apoptosis, indicating that the mineralocorticoid receptor (MR) plays a role. SP-600125 (3 μM)—a selective inhibitor of c-Jun NH2-terminal kinase (JNK)—inhibited aldosterone-induced apoptosis in ARVM. Although aldosterone increased the expression of both stress-activated protein kinases, pretreatment with fenofibrate (10 μM) decreased aldosterone-mediated apoptosis by inhibiting only JNK phosphorylation and the aldosterone-induced increases in Bax, p53, and cleaved caspase-3 and decreases in Bcl-2 protein expression in ARVM. In vivo studies demonstrated that chronic fenofibrate (100 mg·kg body wt−1·day−1) inhibited myocardial Bax and increased Bcl-2 expression in aldosterone-induced cardiac hypertrophy. Similarly, eplerenone, a selective MR inhibitor, used in chronic pressure-overload ascending aortic constriction inhibited myocardial Bax expression but had no effect on Bcl-2 expression. Therefore, involvement of JNK MAPK-dependent mitochondrial death pathway mediates ARVM aldosterone-induced apoptosis and is inhibited by fenofibrate, a peroxisome proliferator-activated receptor (PPAR)α ligand. Fenofibrate mediates beneficial effects in cardiac remodeling by inhibiting programmed cell death and the stress-activated kinases. PMID:19395558

The PP2C Alphabet is a negative regulator of stress-activated protein kinase signaling in Drosophila.

PubMed

Baril, Caroline; Sahmi, Malha; Ashton-Beaucage, Dariel; Stronach, Beth; Therrien, Marc

2009-02-01

The Jun N-terminal kinase and p38 pathways, also known as stress-activated protein kinase (SAPK) pathways, are signaling conduits reiteratively used throughout the development and adult life of metazoans where they play central roles in the control of apoptosis, immune function, and environmental stress responses. We recently identified a Drosophila Ser/Thr phosphatase of the PP2C family, named Alphabet (Alph), which acts as a negative regulator of the Ras/ERK pathway. Here we show that Alph also plays an inhibitory role with respect to Drosophila SAPK signaling during development as well as under stress conditions such as oxidative or genotoxic stresses. Epistasis experiments suggest that Alph acts at a step upstream of the MAPKKs Hep and Lic. Consistent with this interpretation, biochemical experiments identify the upstream MAPKKKs Slpr, Tak1, and Wnd as putative substrates. Together with previous findings, this work identifies Alph as a general attenuator of MAPK signaling in Drosophila.

N-terminal deletions in Rous sarcoma virus p60src: effects on tyrosine kinase and biological activities and on recombination in tissue culture with the cellular src gene.

PubMed Central

Cross, F R; Garber, E A; Hanafusa, H

1985-01-01

We have constructed deletions within the region of cloned Rous sarcoma virus DNA coding for the N-terminal 30 kilodaltons of p60src. Infectious virus was recovered after transfection. Deletions of amino acids 15 to 149, 15 to 169, or 149 to 169 attenuated but did not abolish transforming activity, as assayed by focus formation and anchorage-independent growth. These deletions also had only slight effects on the tyrosine kinase activity of the mutant src protein. Deletion of amino acids 169 to 264 or 15 to 264 completely abolished transforming activity, and src kinase activity was reduced at least 10-fold. However, these mutant viruses generated low levels of transforming virus by recombination with the cellular src gene. The results suggest that as well as previously identified functional domains for p60src myristylation and membrane binding (amino acids 1 to 14) and tyrosine kinase activity (amino acids 250 to 526), additional N-terminal sequences (particularly amino acids 82 to 169) can influence the transforming activity of the src protein. Images PMID:2426576

JNK-Interacting Protein 3 Mediates the Retrograde Transport of Activated c-Jun N-Terminal Kinase and Lysosomes

PubMed Central

Drerup, Catherine M.; Nechiporuk, Alex V.

2013-01-01

Retrograde axonal transport requires an intricate interaction between the dynein motor and its cargo. What mediates this interaction is largely unknown. Using forward genetics and a novel in vivo imaging approach, we identified JNK-interacting protein 3 (Jip3) as a direct mediator of dynein-based retrograde transport of activated (phosphorylated) c-Jun N-terminal Kinase (JNK) and lysosomes. Zebrafish jip3 mutants (jip3nl7) displayed large axon terminal swellings that contained high levels of activated JNK and lysosomes, but not other retrograde cargos such as late endosomes and autophagosomes. Using in vivo analysis of axonal transport, we demonstrated that the terminal accumulations of activated JNK and lysosomes were due to a decreased frequency of retrograde movement of these cargos in jip3nl7, whereas anterograde transport was largely unaffected. Through rescue experiments with Jip3 engineered to lack the JNK binding domain and exogenous expression of constitutively active JNK, we further showed that loss of Jip3–JNK interaction underlies deficits in pJNK retrograde transport, which subsequently caused axon terminal swellings but not lysosome accumulation. Lysosome accumulation, rather, resulted from loss of lysosome association with dynein light intermediate chain (dynein accessory protein) in jip3nl7, as demonstrated by our co-transport analyses. Thus, our results demonstrate that Jip3 is necessary for the retrograde transport of two distinct cargos, active JNK and lysosomes. Furthermore, our data provide strong evidence that Jip3 in fact serves as an adapter protein linking these cargos to dynein. PMID:23468645

The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity.

PubMed

Ando, Maya; Fiesel, Fabienne C; Hudec, Roman; Caulfield, Thomas R; Ogaki, Kotaro; Górka-Skoczylas, Paulina; Koziorowski, Dariusz; Friedman, Andrzej; Chen, Li; Dawson, Valina L; Dawson, Ted M; Bu, Guojun; Ross, Owen A; Wszolek, Zbigniew K; Springer, Wolfdieter

2017-04-24

Mutations in PINK1 and PARKIN are the most common causes of recessive early-onset Parkinson's disease (EOPD). Together, the mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PARKIN direct a complex regulated, sequential mitochondrial quality control. Thereby, damaged mitochondria are identified and targeted to degradation in order to prevent their accumulation and eventually cell death. Homozygous or compound heterozygous loss of either gene function disrupts this protective pathway, though at different steps and by distinct mechanisms. While structure and function of PARKIN variants have been well studied, PINK1 mutations remain poorly characterized, in particular under endogenous conditions. A better understanding of the exact molecular pathogenic mechanisms underlying the pathogenicity is crucial for rational drug design in the future. Here, we characterized the pathogenicity of the PINK1 p.I368N mutation on the clinical and genetic as well as on the structural and functional level in patients' fibroblasts and in cell-based, biochemical assays. Under endogenous conditions, PINK1 p.I368N is expressed, imported, and N-terminally processed in healthy mitochondria similar to PINK1 wild type (WT). Upon mitochondrial damage, however, full-length PINK1 p.I368N is not sufficiently stabilized on the outer mitochondrial membrane (OMM) resulting in loss of mitochondrial quality control. We found that binding of PINK1 p.I368N to the co-chaperone complex HSP90/CDC37 is reduced and stress-induced interaction with TOM40 of the mitochondrial protein import machinery is abolished. Analysis of a structural PINK1 p.I368N model additionally suggested impairments of Ub kinase activity as the ATP-binding pocket was found deformed and the substrate Ub was slightly misaligned within the active site of the kinase. Functional assays confirmed the lack of Ub kinase activity. Here we demonstrated that mutant PINK1 p.I368N can not be stabilized on the OMM upon

Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response

PubMed Central

Chan, Tung O.; Zhang, Jin; Tiegs, Brian C.; Blumhof, Brian; Yan, Linda; Keny, Nikhil; Penny, Morgan; Li, Xue; Pascal, John M.; Armen, Roger S.; Rodeck, Ulrich; Penn, Raymond B.

2015-01-01

The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr308 in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr308 dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser473) increased phosphatase resistance of the phosphorylated activation loop (pThr308) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr308 phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin. PMID:26201515

Activation of c-jun N-terminal kinase upon influenza A virus (IAV) infection is independent of pathogen-related receptors but dependent on amino acid sequence variations of IAV NS1.

PubMed

Nacken, Wolfgang; Anhlan, Darisuren; Hrincius, Eike R; Mostafa, Ahmed; Wolff, Thorsten; Sadewasser, Anne; Pleschka, Stephan; Ehrhardt, Christina; Ludwig, Stephan

2014-08-01

A hallmark cell response to influenza A virus (IAV) infections is the phosphorylation and activation of c-jun N-terminal kinase (JNK). However, so far it is not fully clear which molecules are involved in the activation of JNK upon IAV infection. Here, we report that the transfection of influenza viral-RNA induces JNK in a retinoic acid-inducible gene I (RIG-I)-dependent manner. However, neither RIG-I-like receptors nor MyD88-dependent Toll-like receptors were found to be involved in the activation of JNK upon IAV infection. Viral JNK activation may be blocked by addition of cycloheximide and heat shock protein inhibitors during infection, suggesting that the expression of an IAV-encoded protein is responsible for JNK activation. Indeed, the overexpression of nonstructural protein 1 (NS1) of certain IAV subtypes activated JNK, whereas those of some other subtypes failed to activate JNK. Site-directed mutagenesis experiments using NS1 of the IAV H7N7, H5N1, and H3N2 subtypes identified the amino acid residue phenylalanine (F) at position 103 to be decisive for JNK activation. Cleavage- and polyadenylation-specific factor 30 (CPSF30), whose binding to NS1 is stabilized by the amino acids F103 and M106, is not involved in JNK activation. Conclusively, subtype-specific sequence variations in the IAV NS1 protein result in subtype-specific differences in JNK signaling upon IAV infection. Influenza A virus (IAV) infection leads to the activation or modulation of multiple signaling pathways. Here, we demonstrate for the first time that the c-jun N-terminal kinase (JNK), a long-known stress-activated mitogen-activated protein (MAP) kinase, is activated by RIG-I when cells are treated with IAV RNA. However, at the same time, nonstructural protein 1 (NS1) of IAV has an intrinsic JNK-activating property that is dependent on IAV subtype-specific amino acid variations around position 103. Our findings identify two different and independent pathways that result in the activation

Activation of c-jun N-Terminal Kinase upon Influenza A Virus (IAV) Infection Is Independent of Pathogen-Related Receptors but Dependent on Amino Acid Sequence Variations of IAV NS1

PubMed Central

Nacken, Wolfgang; Anhlan, Darisuren; Hrincius, Eike R.; Mostafa, Ahmed; Wolff, Thorsten; Sadewasser, Anne; Pleschka, Stephan; Ehrhardt, Christina

2014-01-01

ABSTRACT A hallmark cell response to influenza A virus (IAV) infections is the phosphorylation and activation of c-jun N-terminal kinase (JNK). However, so far it is not fully clear which molecules are involved in the activation of JNK upon IAV infection. Here, we report that the transfection of influenza viral-RNA induces JNK in a retinoic acid-inducible gene I (RIG-I)-dependent manner. However, neither RIG-I-like receptors nor MyD88-dependent Toll-like receptors were found to be involved in the activation of JNK upon IAV infection. Viral JNK activation may be blocked by addition of cycloheximide and heat shock protein inhibitors during infection, suggesting that the expression of an IAV-encoded protein is responsible for JNK activation. Indeed, the overexpression of nonstructural protein 1 (NS1) of certain IAV subtypes activated JNK, whereas those of some other subtypes failed to activate JNK. Site-directed mutagenesis experiments using NS1 of the IAV H7N7, H5N1, and H3N2 subtypes identified the amino acid residue phenylalanine (F) at position 103 to be decisive for JNK activation. Cleavage- and polyadenylation-specific factor 30 (CPSF30), whose binding to NS1 is stabilized by the amino acids F103 and M106, is not involved in JNK activation. Conclusively, subtype-specific sequence variations in the IAV NS1 protein result in subtype-specific differences in JNK signaling upon IAV infection. IMPORTANCE Influenza A virus (IAV) infection leads to the activation or modulation of multiple signaling pathways. Here, we demonstrate for the first time that the c-jun N-terminal kinase (JNK), a long-known stress-activated mitogen-activated protein (MAP) kinase, is activated by RIG-I when cells are treated with IAV RNA. However, at the same time, nonstructural protein 1 (NS1) of IAV has an intrinsic JNK-activating property that is dependent on IAV subtype-specific amino acid variations around position 103. Our findings identify two different and independent pathways that

Lower Susceptibility of Female Mice to Acetaminophen Hepatotoxicity: Role of Mitochondrial Glutathione, Oxidant Stress and c-Jun N-Terminal Kinase

PubMed Central

Du, Kuo; Williams, C. David; McGill, Mitchell R.; Jaeschke, Hartmut

2014-01-01

Acetaminophen (APAP) overdose causes severe hepatotoxicity in animals and humans. However, the mechanisms underlying the gender differences in susceptibility to APAP overdose in mice have not been clarified. In our study, APAP (300 mg/kg) caused severe liver injury in male mice but 69-77% lower injury in females. No gender difference in metabolic activation of APAP was found. Hepatic glutathione (GSH) was rapidly depleted in both genders, while GSH recovery in female mice was 2.6 fold higher in mitochondria at 4h, and 2.5 and 3.3 fold higher in the total liver at 4h and 6h, respectively. This faster recovery of GSH, which correlated with greater induction of glutamate-cysteine ligase, attenuated mitochondrial oxidative stress in female mice, as suggested by a lower GSSG/GSH ratio at 6h (3.8% in males vs. 1.4% in females) and minimal centrilobular nitrotyrosine staining. While c-jun N-terminal kinase (JNK) activation was similar at 2 and 4h post-APAP, it was 3.1 fold lower at 6h in female mice. However, female mice were still protected by the JNK inhibitor SP600125. 17β-Estradiol pretreatment moderately decreased liver injury and oxidative stress in male mice without affecting GSH recovery. Conclusion: The lower susceptibility of female mice is achieved by the improved detoxification of reactive oxygen due to accelerated recovery of mitochondrial GSH levels, which attenuates late JNK activation and liver injury. However, even the reduced injury in female mice was still dependent on JNK. While 17β-estradiol partially protects male mice, it does not affect hepatic GSH recovery. PMID:25218290

c-Jun N-terminal kinase modulates oxidant stress and peroxynitrite formation independent of inducible nitric oxide synthase in acetaminophen hepatotoxicity

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

Saito, Chieko; Lemasters, John J.; Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425

Acetaminophen (APAP) overdose, which causes liver injury in animals and humans, activates c-jun N-terminal kinase (JNK). Although it was shown that the JNK inhibitor SP600125 effectively reduced APAP hepatotoxicity, the mechanisms of protection remain unclear. C57Bl/6 mice were treated with 10 mg/kg SP600125 or vehicle (8% dimethylsulfoxide) 1 h before 600 mg/kg APAP administration. APAP time-dependently induced JNK activation (detected by JNK phosphorylation). SP600125, but not the vehicle, reduced JNK activation, attenuated mitochondrial Bax translocation and prevented the mitochondrial release of apoptosis-inducing factor at 4-12 h. Nuclear DNA fragmentation, nitrotyrosine staining, tissue GSSG levels and liver injury (plasma ALT releasemore » and necrosis) were partially attenuated by the vehicle (- 65%) and completely eliminated by SP600125 (- 98%) at 6 and 12 h. Furthermore, SP600125 attenuated the increase of inducible nitric oxide synthase (iNOS) mRNA and protein. However, APAP did not enhance plasma nitrite + nitrate levels (NO formation); SP600125 had no effect on this parameter. The iNOS inhibitor L-NIL did not reduce NO formation or injury after APAP but prevented NO formation caused by endotoxin. Since SP600125 completely eliminated the increase in hepatic GSSG levels, an indicator of mitochondrial oxidant stress, it is concluded that the inhibition of peroxynitrite was mainly caused by reduced superoxide formation. Our data suggest that the JNK inhibitor SP600125 protects against APAP-induced liver injury in part by attenuation of mitochondrial Bax translocation but mainly by preventing mitochondrial oxidant stress and peroxynitrite formation and thereby preventing the mitochondrial permeability transition pore opening, a key event in APAP-induced cell necrosis.« less

Lower susceptibility of female mice to acetaminophen hepatotoxicity: Role of mitochondrial glutathione, oxidant stress and c-jun N-terminal kinase

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

Du, Kuo; Williams, C. David; McGill, Mitchell R.

Acetaminophen (APAP) overdose causes severe hepatotoxicity in animals and humans. However, the mechanisms underlying the gender differences in susceptibility to APAP overdose in mice have not been clarified. In our study, APAP (300 mg/kg) caused severe liver injury in male mice but 69–77% lower injury in females. No gender difference in metabolic activation of APAP was found. Hepatic glutathione (GSH) was rapidly depleted in both genders, while GSH recovery in female mice was 2.6 fold higher in the mitochondria at 4 h, and 2.5 and 3.3 fold higher in the total liver at 4 h and 6 h, respectively. Thismore » faster recovery of GSH, which correlated with greater induction of glutamate-cysteine ligase, attenuated mitochondrial oxidative stress in female mice, as suggested by a lower GSSG/GSH ratio at 6 h (3.8% in males vs. 1.4% in females) and minimal centrilobular nitrotyrosine staining. While c-jun N-terminal kinase (JNK) activation was similar at 2 and 4 h post-APAP, it was 3.1 fold lower at 6 h in female mice. However, female mice were still protected by the JNK inhibitor SP600125. 17β-Estradiol pretreatment moderately decreased liver injury and oxidative stress in male mice without affecting GSH recovery. Conclusion: The lower susceptibility of female mice is achieved by the improved detoxification of reactive oxygen due to accelerated recovery of mitochondrial GSH levels, which attenuates late JNK activation and liver injury. However, even the reduced injury in female mice was still dependent on JNK. While 17β-estradiol partially protects male mice, it does not affect hepatic GSH recovery. - Highlights: • Female mice are less susceptible to acetaminophen overdose than males. • GSH depletion and protein adduct formation are similar in both genders. • Recovery of hepatic GSH levels is faster in females and correlates with Gclc. • Reduced oxidant stress in females leads to reduced JNK activation. • JNK activation and mitochondrial translocation are

Protective role of c-Jun N-terminal kinase 2 in acetaminophen-induced liver injury

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

Bourdi, Mohammed; Korrapati, Midhun C.; Chakraborty, Mala

2008-09-12

Recent studies in mice suggest that stress-activated c-Jun N-terminal protein kinase 2 (JNK2) plays a pathologic role in acetaminophen (APAP)-induced liver injury (AILI), a major cause of acute liver failure (ALF). In contrast, we present evidence that JNK2 can have a protective role against AILI. When male C57BL/6J wild type (WT) and JNK2{sup -/-} mice were treated with 300 mg APAP/kg, 90% of JNK2{sup -/-} mice died of ALF compared to 20% of WT mice within 48 h. The high susceptibility of JNK2{sup -/-} mice to AILI appears to be due in part to deficiencies in hepatocyte proliferation and repair.more » Therefore, our findings are consistent with JNK2 signaling playing a protective role in AILI and further suggest that the use of JNK inhibitors as a potential treatment for AILI, as has been recommended by other investigators, should be reconsidered.« less

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

PubMed Central

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

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

PubMed

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.

Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response.

PubMed

Chan, Tung O; Zhang, Jin; Tiegs, Brian C; Blumhof, Brian; Yan, Linda; Keny, Nikhil; Penny, Morgan; Li, Xue; Pascal, John M; Armen, Roger S; Rodeck, Ulrich; Penn, Raymond B

2015-10-01

The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr(308) in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr(308) dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser(473)) increased phosphatase resistance of the phosphorylated activation loop (pThr(308)) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr(308) phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin. © 2015 Authors; published by Portland Press Limited.

Emerging role of the Jun N-terminal kinase interactome in human health.

PubMed

Guo, Xiao-Xi; An, Su; Yang, Yang; Liu, Ying; Hao, Qian; Tang, Tao; Xu, Tian-Rui

2018-02-08

The c-Jun N-terminal kinases (JNKs) are located downstream of Ras-mitogen activated protein kinase signaling cascades. More than 20 years of study has shown that JNKs control cell fate and many cellular functions. JNKs and their interacting proteins form a complicated network with diverse biological functions and physiological effects. Members of the JNK interactome include Jun, amyloid precursor protein, and insulin receptor substrate. Recent studies have shown that the JNK interactome is involved in tumorigenesis, neuron development, and insulin resistance. In this review, we summarize the features of the JNK interactome and classify its members into three groups: upstream regulators, downstream effectors, and scaffold partners. We also highlight the unique cellular signaling mechanisms of JNKs and provide more insights into the roles of the JNK interactome in human diseases. © 2018 International Federation for Cell Biology.

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

PubMed Central

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

2017-01-01

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

Mutant human tumor suppressor p53 modulates the activation of mitogen-activated protein kinase and nuclear factor-kappaB, but not c-Jun N-terminal kinase and activated protein-1.

PubMed

Gulati, Anthony P; Yang, Yang-Ming; Harter, David; Mukhopadhyay, Asok; Aggarwal, Bharat B; Aggarwal, Bharat A; Benzil, Deborah L; Whysner, John; Albino, Anthony P; Murali, Raj; Jhanwar-Uniyal, Meena

2006-01-01

The roles of the mitogen-activated kinase protein (MAPK) pathway, nuclear factor-kappa B (NF-kappaB), and activator protein-1 (AP-1) in cellular responses to growth factors and mitogen are well established. However, the manner by which these proliferative pathways are affected by the tumor suppressor protein p53 is not fully understood. We report here the results of an investigation of the status of p53 on two human melanoma cell lines with wild-type p53 (SK-Mel-186) or mutant p53 (SK-Mel-110). The basal levels of the activated extracellular-signal regulated kinases 1 and 2 (ERK1/2) were high in cells with wild-type p53, but low in cells with mutant p53. The 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of ERK1/2 through the phosphorylation of threonine and tyrosine at 202 and 204, respectively, was demonstrated in both cell lines, however, in a discrete manner. TPA-induced activation of ERK1/2 was sustained in wild-type p53 cells, while only a transient activation was seen in mutant p53 cells. Inhibition of MAPK kinase (MEK), an upstream kinase, by U0126, blocked TPA-induced activation of ERK1/2 in wild-type p53 cells and in mutant p53 cells. Treatment of wild-type p53 (SK-Mel 186) cells with small interfering RNA (siRNA) of p53 displayed a transient induction of activation of ERK1/2 following TPA treatment, indicating that p53 has a role in the regulation of the activation of ERK1/2. NF-kappaB activity decreased significantly in cells with wild-type p53, while enhanced NF-kappaB activity was evident in cells with mutant p53. The expression of either wild-type or mutant p53 had a similar effect on TPA-induced Jun N-terminal kinase (JNK) activation, indicating specificity for the ERK pathway. Similarly, AP-1 binding activity showed a transient variation in both cell lines after TPA treatment but with different kinetics. These observations suggest that both wild-type and mutant p53 can modulate the activation pathways for ERK1/2, and NF

An N-terminally truncated form of cyclic GMP-dependent protein kinase Iα (PKG Iα) is monomeric and autoinhibited and provides a model for activation.

PubMed

Moon, Thomas M; Sheehe, Jessica L; Nukareddy, Praveena; Nausch, Lydia W; Wohlfahrt, Jessica; Matthews, Dwight E; Blumenthal, Donald K; Dostmann, Wolfgang R

2018-05-25

The type I cGMP-dependent protein kinases (PKG I) serve essential physiological functions, including smooth muscle relaxation, cardiac remodeling, and platelet aggregation. These enzymes form homodimers through their N-terminal dimerization domains, a feature implicated in regulating their cooperative activation. Previous investigations into the activation mechanisms of PKG I isoforms have been largely influenced by structures of the cAMP-dependent protein kinase (PKA). Here, we examined PKG Iα activation by cGMP and cAMP by engineering a monomeric form that lacks N-terminal residues 1-53 (Δ53). We found that the construct exists as a monomer as assessed by whole-protein MS, size-exclusion chromatography, and small-angle X-ray scattering (SAXS). Reconstruction of the SAXS 3D envelope indicates that Δ53 has a similar shape to the heterodimeric RIα-C complex of PKA. Moreover, we found that the Δ53 construct is autoinhibited in its cGMP-free state and can bind to and be activated by cGMP in a manner similar to full-length PKG Iα as assessed by surface plasmon resonance (SPR) spectroscopy. However, we found that the Δ53 variant does not exhibit cooperative activation, and its cyclic nucleotide selectivity is diminished. These findings support a model in which, despite structural similarities, PKG Iα activation is distinct from that of PKA, and its cooperativity is driven by in trans interactions between protomers. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed Central

2012-01-01

Background Based on an experimental brain stem death model, we demonstrated previously that activation of the mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ mitogen-activated protein kinase signal-interacting kinase 1/2 (MNK1/2) cascade plays a pro-life role in the rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life) and decreases (pro-death) to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients. The present study assessed the hypothesis that, in addition to ERK1/2, c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), the other two mammalian members of MAPKs that are originally identified as stress-activated protein kinases, are activated specifically by MAPK kinase 4 (MAP2K4) or MAP2K6 and play a pro-life role in RVLM during experimental brain stem death. We further delineated the participation of phosphorylating activating transcriptional factor-2 (ATF-2) and c-Jun, the classical transcription factor activated by JNK or p38MAPK, in this process. Results An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol) bilaterally into RVLM of Sprague–Dawley rats was used, alongside cardiovascular, pharmacological and biochemical evaluations. Results from ELISA showed that whereas the total JNK, p38MAPK, MAP2K4 and MAP2K6 were not affected, augmented phosphorylation of JNK at Thr183 and Tyr185 and p38MAPK at Thr180 and Tyr182, accompanied by phosphorylation of their upstream activators MAP2K4 at Ser257 and Thr261 and MAP2K6 at Ser207 and Thr211 in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Moreover, the activity of transcription factors ATF-2 at Thr71 and c-Jun at Ser73

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

PubMed

Chang, Alice Y W

2012-11-17

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

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

PubMed

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

1998-08-15

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

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

PubMed Central

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

1998-01-01

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

Human Protein Kinases and Obesity.

PubMed

Engin, Atilla

2017-01-01

The action of protein kinases and protein phosphatases is essential for multiple physiological responses. Each protein kinase displays its own unique substrate specificity, and a regulatory mechanism that may be modulated by association with other proteins. Protein kinases are classified by the target amino acid in their substrates. Some protein kinases can phosphorylate both serine/threonine, as well as tyrosine residues. This group of kinases has been known as dual specificity kinases. Unlike the dual specificity kinases, a heterogeneous group of protein phosphatases are known as dual-specificity phosphatases. These phosphatases remove phosphate groups from tyrosine and serine/threonine residues on their substrate. Dual-specificity phosphatases are important signal transduction enzymes that regulate various cellular processes in coordination with protein kinases. The protein kinase-phosphoproteins interactions play an important role in obesity . In obesity, the pro- and anti-inflammatory effects of adipokines and cytokines through intracellular signaling pathways mainly involve the nuclear factor kappa B (NF-kappaB) and the c-Jun N-terminal kinase (JNK) systems as well as the inhibitor of kappaB-kinase beta (IKK beta). Impairment of insulin signaling in obesity is largely mediated by the activation of the IKKbeta and the JNK. Furthermore, oxidative stress and endoplasmic reticulum (ER) stress activate the JNK pathway which suppresses insulin biosynthesis. Additionally, obesity-activated calcium/calmodulin dependent-protein kinase II/p38 suppresses insulin-induced protein kinase B phosphorylation by activating the ER stress effector, activating transcription factor-4. Obese adults with vascular endothelial dysfunction have greater endothelial cells activation of unfolded protein response stress sensors, RNA-dependent protein kinase-like ER eukaryotic initiation factor-2alpha kinase (PERK) and activating transcription factor-6. The transcriptional regulation of

Auto-phosphorylation Represses Protein Kinase R Activity.

PubMed

Wang, Die; de Weerd, Nicole A; Willard, Belinda; Polekhina, Galina; Williams, Bryan R G; Sadler, Anthony J

2017-03-10

The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity.

Trovafloxacin potentiation of lipopolysaccharide-induced tumor necrosis factor release from RAW 264.7 cells requires extracellular signal-regulated kinase and c-Jun N-Terminal Kinase.

PubMed

Poulsen, Kyle L; Albee, Ryan P; Ganey, Patricia E; Roth, Robert A

2014-05-01

Trovafloxacin (TVX) is a fluoroquinolone antibiotic known to cause idiosyncratic, drug-induced liver injury (IDILI) in humans. The mechanism underlying this toxicity remains unknown. Previously, an animal model of IDILI in mice revealed that TVX synergizes with inflammatory stress from bacterial lipopolysaccharide (LPS) to produce a hepatotoxic interaction. The liver injury required prolongation of the appearance of tumor necrosis factor-α (TNF) in the plasma. The results presented here describe a model of TVX/LPS coexposure in RAW 264.7 cells acting as a surrogate for TNF-releasing cells in vivo. Pretreating cells with TVX for 2 hours before LPS addition led to increased TNF protein release into culture medium in a concentration- and time-dependent manner relative to cells treated with LPS or TVX alone. During the pretreatment period, TVX increased TNF mRNA, but this was less apparent when cells were exposed to TVX after LPS addition, suggesting that the pivotal signaling events that increase TNF expression occurred during the TVX pretreatment period. Indeed, TVX exposure increased activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase. Inhibition of either ERK or JNK decreased the TVX-mediated increase in TNF mRNA and LPS-induced TNF protein release, but p38 inhibition did not. These results demonstrated that the increased TNF appearance from TVX-LPS interaction in vivo can be reproduced in vitro and occurs in an ERK- and JNK-dependent manner.

Human Nek6 is a monomeric mostly globular kinase with an unfolded short N-terminal domain

PubMed Central

2011-01-01

Background The NIMA-related kinases (Neks) are widespread among eukaryotes. In mammalians they represent an evolutionarily conserved family of 11 serine/threonine kinases, with 40-45% amino acid sequence identity to the Aspergillus nidulans mitotic regulator NIMA within their catalytic domains. Neks have cell cycle-related functions and were recently described as related to pathologies, particularly cancer, consisting in potential chemotherapeutic targets. Human Nek6, -7 and -9 are involved in the control of mitotic spindle formation, acting together in a mitotic kinase cascade, but their mechanism of regulation remain elusive. Results In this study we performed a biophysical and structural characterization of human Nek6 with the aim of obtaining its low resolution and homology models. SAXS experiments showed that hNek6 is a monomer of a mostly globular, though slightly elongated shape. Comparative molecular modeling together with disorder prediction analysis also revealed a flexible disordered N-terminal domain for hNek6, which we found to be important to mediate interactions with diverse partners. SEC-MALS experiments showed that hNek6 conformation is dependent on its activation/phosphorylation status, a higher phosphorylation degree corresponding to a bigger Stokes radius. Circular dichroism spectroscopy confirmed our in silico predictions of secondary structure content and thermal stability shift assays revealed a slightly higher stability of wild-type hNek6 compared to the activation loop mutant hNek6(S206A). Conclusions Our data present the first low resolution 3D structure of hNek6 protein in solution. SAXS, comparative modeling and SEC-MALS analysis revealed that hNek6 is a monomeric kinase of slightly elongated shape and a short unfolded N-terminal domain. PMID:21320329

Arrestin-3-dependent activation of c-Jun N-terminal kinases (JNKs)

PubMed Central

Kaoud, Tamer S.; Dalby, Kevin N.; Gurevich, Eugenia V.; Gurevich, Vsevolod V.

2015-01-01

Only one out of four arrestin subtypes expressed in mammals, arrestin-3, facilitates the activation of JNK family kinases. Here we describe two different paradigms that allow the elucidation of the mechanisms involved. One is based on reconstitution of signaling modules from purified proteins: arrestin-3, MKK4, MKK7, JNK1, JNK2, and JNK3. The main advantage of this method is that it can unambiguously establish which effects are direct, because only intended purified proteins are present in these assays. The key drawback is that the upstream-most kinases of these cascades, ASK1 or other MAPKKKs, are not available in purified form, limiting reconstitution to incomplete two-kinase modules. The other set of methods analyzes the effects of arrestin-3 on JNK activation in intact cells. In this case, signaling modules include ASK1 and/or other MAPKKKs. However, every cell expresses thousands of different proteins, and their possible effects on the readout cannot be excluded. However, the combination of in vitro reconstitution from purified proteins and cell-based assays enables comprehensive elucidation of the mechanisms of arrestin-3-dependent activation of JNK family kinases. PMID:25737158

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

PubMed

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

2000-12-01

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

Ketamine inhibits tumor necrosis factor-{alpha} and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation

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

Wu, G.-J.; Department of Anesthesiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

2008-04-01

Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-{alpha} (TNF-{alpha}) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 {mu}M ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 {mu}M of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-{alpha}more » and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-{alpha} and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 {mu}M) significantly inhibited LPS-induced TNF-{alpha} and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-{alpha} and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-{alpha} and IL-6 gene expressions in LPS-activated macrophages. The suppressive

Ketamine inhibits tumor necrosis factor-alpha and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation.

PubMed

Wu, Gone-Jhe; Chen, Ta-Liang; Ueng, Yune-Fang; Chen, Ruei-Ming

2008-04-01

Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-alpha (TNF-alpha) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 microM ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 microM of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-alpha and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-alpha and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 microM) significantly inhibited LPS-induced TNF-alpha and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-alpha and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-alpha and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through

Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

PubMed

Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Sun, Kaiji; Zhang, Qing; Wu, Guoyao

2016-01-01

High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P < 0.05) and led to vacuole-like cell death in intestinal porcine epithelial cells. These adverse effects of L-cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P < 0.05), whereas those for p-ERK1/2 were reduced (P < 0.05). Collectively, excessive L-cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

Dexamethasone inhibits IL-12p40 production in lipopolysaccharide-stimulated human monocytic cells by down-regulating the activity of c-Jun N-terminal kinase, the activation protein-1, and NF-kappa B transcription factors.

PubMed

Ma, Wei; Gee, Katrina; Lim, Wilfred; Chambers, Kelly; Angel, Jonathan B; Kozlowski, Maya; Kumar, Ashok

2004-01-01

IL-12 plays a critical role in the development of cell-mediated immune responses and in the pathogenesis of inflammatory and autoimmune disorders. Dexamethasone (DXM), an anti-inflammatory glucocorticoid, has been shown to inhibit IL-12p40 production in LPS-stimulated monocytic cells. In this study, we investigated the molecular mechanism by which DXM inhibits IL-12p40 production by studying the role of the mitogen-activated protein kinases (MAPKs), and the key transcription factors involved in human IL-12p40 production in LPS-stimulated monocytic cells. A role for c-Jun N-terminal kinase (JNK) MAPK in LPS-induced IL-12p40 regulation in a promonocytic THP-1/CD14 cell line was demonstrated by using specific inhibitors of JNK activation, SP600125 and a dominant-negative stress-activated protein/extracellular signal-regulated kinase kinase-1 mutant. To identify transcription factors regulating IL-12p40 gene transcription, extensive deletion analyses of the IL-12p40 promoter was performed. The results revealed the involvement of a sequence encompassing the AP-1-binding site, in addition to that of NF-kappaB. The role of AP-1 in IL-12p40 transcription was confirmed by using antisense c-fos and c-jun oligonucleotides. Studies conducted to understand the regulation of AP-1 and NF-kappaB activation by JNK MAPK revealed that both DXM and SP600125 inhibited IL-12p40 gene transcription by inhibiting the activation of AP-1 and NF-kappaB transcription factors as revealed by luciferase reporter and gel mobility shift assays. Taken together, our results suggest that DXM may inhibit IL-12p40 production in LPS-stimulated human monocytic cells by down-regulating the activation of JNK MAPK, the AP-1, and NF-kappaB transcription factors.

Methotrexate increases expression of cell cycle checkpoint genes via Jun-N-terminal kinase activation

PubMed Central

Spurlock, Charles F.; Tossberg, John T.; Fuchs, Howard A.; Olsen, Nancy J.; Aune, Thomas M.

2011-01-01

Objective To assess defects in expression of critical cell cycle checkpoint genes and proteins in subjects with rheumatoid arthritis relative to presence or absence of methotrexate medication and assess the role of Jun N-terminal kinase in methotrexate induction of these genes. Methods Flow cytometry analysis was used to quantify changes in intracellular proteins, measure reactive oxygen species (ROS), and determine apoptosis in different lymphoid populations. Quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) was employed to determine changes in cell cycle checkpoint target genes. Results RA subjects express lower baseline levels of MAPK9, TP53, CDKN1A, CDKN1B, CHEK2, and RANGAP1 messenger RNA (mRNA) and total JNK protein. MAPK9, TP53, CDKN1A, and CDKN1B mRNA expression, but not CHEK2, and RANGAP1, is higher in patients on low-dose MTX therapy. Further, JNK levels inversely correlate with CRP levels in RA patients. In tissue culture, MTX induces expression of both p53 and p21 by JNK2 and JNK1-dependent mechanisms, respectively, while CHEK2 and RANGAP1 are not induced by MTX. MTX also induces ROS production, JNK activation, and sensitivity to apoptosis in activated T cells. Supplementation with tetrahydrobiopterin blocks these MTX-mediated effects. Conclusions Our findings support the notion that MTX restores some, but not all of the proteins contributing to cell cycle checkpoint deficiencies in RA T cells by a JNK dependent pathway. PMID:22183962

The activation of c-Jun NH2-terminal kinase is required for dihydroartemisinin-induced autophagy in pancreatic cancer cells

PubMed Central

2014-01-01

Background c-Jun NH2-terminal kinases (JNKs) are strongly activated by a stressful cellular environment, such as chemotherapy and oxidative stress. Autophagy is a protein-degradation system in which double-membrane vacuoles called autophagosomes are formed. The autophagy-related gene Beclin 1 plays a key role in this process. We previously found that autophagy was induced by dihydroartemisinin (DHA) in pancreatic cancer cells. However, little is known about the complex relationship between ROS, JNK activation, autophagy induction, and Beclin 1 expression. Methods Cell viability and CCK-8 assays were carried out to determine the cell proliferation; small interfering RNAs (siRNAs) were used to knockdown c-Jun NH2-terminal kinases (JNK1/2) genes; western blot was performed to detect the protein expression of LC3, JNK, Beclin 1, caspase 3 and β-actin; production of intracellular ROS was analyzed using FACS flow cytometry; autophagy induction was confirmed by electron microscopy. Results In the present study, we explored the role of DHA and Beclin 1 expression in autophagy. DHA-treated cells showed autophagy characteristics, and DHA also activated the JNK pathway and up-regulated the expression of Beclin 1. Conversely, blocking JNK signaling inhibited Beclin 1 up-regulation. JNK activation was found to primarily depend on reactive oxygen species (ROS) resulting from the DHA treatment. Moreover, JNK pathway inhibition and Beclin 1 silencing prevented the induction of DHA-induced autophagy. Conclusions These results suggest that the induction of autophagy by DHA is required for JNK-mediated Beclin 1 expression. PMID:24438216

Reciprocal signals between microglia and neurons regulate α-synuclein secretion by exophagy through a neuronal cJUN-N-terminal kinase-signaling axis.

PubMed

Christensen, Dan Ploug; Ejlerskov, Patrick; Rasmussen, Izabela; Vilhardt, Frederik

2016-03-08

Secretion of proteopathic α-synuclein (α-SNC) species from neurons is a suspected driving force in the propagation of Parkinson's disease (PD). We have previously implicated exophagy, the exocytosis of autophagosomes, as a dominant mechanism of α-SNC secretion in differentiated PC12 or SH-SY5Y nerve cells. Here we have examined the regulation of exophagy associated with different forms of nerve cell stress relevant to PD. We identify cJUN-N-terminal kinase (JNK) activity as pivotal in the secretory fate of autophagosomes containing α-SNC. Pharmacological inhibition or genetic (shRNA) knockdown of JNK2 or JNK3 decreases α-SNC secretion in differentiated PC12 and SH-SY5Y cells, respectively. Conversely, expression of constitutively active mitogen-activated protein kinase kinase 7 (MKK7)-JNK2 and -JNK3 constructs augment secretion. The transcriptional activity of cJUN was not required for the observed effects. We establish a causal relationship between increased α-SNC release by exophagy and JNK activation subsequent to lysosomal fusion deficiency (overexpression of Lewy body-localized protein p25α or bafilomycin A1). JNK activation following neuronal ER or oxidative stress was not correlated with exophagy, but of note, we demonstrate that reciprocal signaling between microglia and neurons modulates α-SNC secretion. NADPH oxidase activity of microglia cell lines was upregulated by direct co-culture with α-SNC-expressing PC12 neurons or by passive transfer of nerve cell-conditioned medium. Conversely, inflammatory factors secreted from activated microglia increased JNK activation and α-SNC secretion several-fold in PC12 cells. While we do not identify these factors, we extend our observations by showing that exposure of neurons in monoculture to TNFα, a classical pro-inflammatory mediator of activated microglia, is sufficient to increase α-SNC secretion in a mechanism dependent on JNK2 or JNK3. In continuation hereof, we show that also IFNβ and TGF�

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

PubMed

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.

Differential activation of eIF2 kinases in response to cellular stresses in Schizosaccharomyces pombe.

PubMed

Zhan, Ke; Narasimhan, Jana; Wek, Ronald C

2004-12-01

Phosphorylation of eukaryotic initiation factor-2 (eIF2) is an important mechanism mitigating cellular injury in response to diverse environmental stresses. While all eukaryotic organisms characterized to date contain an eIF2 kinase stress response pathway, the composition of eIF2 kinases differs, with mammals containing four distinct family members and the well-studied lower eukaryote Saccharomyces cerevisiae expressing only a single eIF2 kinase. We are interested in the mechanisms by which multiple eIF2 kinases interface with complex stress signals and elicit response pathways. In this report we find that in addition to two previously described eIF2 kinases related to mammalian HRI, designated Hri1p and Hri2p, the yeast Schizosaccharomyces pombe expresses a third eIF2 kinase, a Gcn2p ortholog. To delineate the roles of each eIF2 kinase, we constructed S. pombe strains expressing only a single eIF2 kinase gene or deleted for the entire eIF2 kinase family. We find that Hri2p is the primary activated eIF2 kinase in response to exposure to heat shock, arsenite, or cadmium. Gcn2p serves as the primary eIF2 kinase induced during a nutrient downshift, treatment with the amino acid biosynthetic inhibitor 3-aminotriazole, or upon exposure to high concentrations of sodium chloride. In one stress example, exposure to H(2)O(2), there is early tandem activation of both Hri2p and Gcn2p. Interestingly, with extended stress conditions there is activation of alternative secondary eIF2 kinases, suggesting that eukaryotes have mechanisms of coordinate activation of eIF2 kinase in their stress remediation responses. Deletion of these eIF2 kinases renders S. pombe more sensitive to many of these stress conditions.

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

NASA Technical Reports Server (NTRS)

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

2000-01-01

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

Identification of N-Terminal Lobe Motifs that Determine the Kinase Activity of the Catalytic Domains and Regulatory Strategies of Src and Csk Protein Tyrosine Kinases†

PubMed Central

Huang, Kezhen; Wang, Yue-Hao; Brown, Alex; Sun, Gongqin

2009-01-01

Csk and Src protein tyrosine kinases are structurally homologous, but use opposite regulatory strategies. The isolated catalytic domain of Csk is intrinsically inactive and is activated by interactions with the regulatory SH3 and SH2 domains, while the isolated catalytic domain of Src is intrinsically active and is suppressed by interactions with the regulatory SH3 and SH2 domains. The structural basis for why one isolated catalytic domain is intrinsically active while the other is inactive is not clear. In this current study, we identify the structural elements in the N-terminal lobe of the catalytic domain that render the Src catalytic domain active. These structural elements include the α-helix C region, a β-turn between the β-4 and β-5 strands, and an Arg residue at the beginning of the catalytic domain. These three motifs interact with each other to activate the Src catalytic domain, but the equivalent motifs in Csk directly interact with the regulatory domains that are important for Csk activation. The Src motifs can be grafted to the Csk catalytic domain to obtain an active Csk catalytic domain. These results, together with available Src and Csk tertiary structures, reveal an important structural switch that determines the kinase activity of a catalytic domain and dictates the regulatory strategy of a kinase. PMID:19244618

c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis

DTIC Science & Technology

2013-10-01

Treatment of Amyotrophic Lateral Sclerosis ” PRINCIPAL INVESTIGATOR: Dr. Philip LoGrasso CONTRACTING ORGANIZATION: The Scripps Research... Lateral Sclerosis ” 5a. CONTRACT NUMBER W81XWH-12-1-0431 5b. GRANT NUMBER W81XWH-12-1-0431 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Philip...Annual 3. DATES COVERED 30September2012-29September2013 4. TITLE AND SUBTITLE “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic

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

PubMed

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.

Synthesis and SAR of piperazine amides as novel c-jun N-terminal kinase (JNK) inhibitors

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

Shin, Youseung; Chen, Weiming; Habel, Jeff

2009-09-14

A novel series of c-jun N-terminal kinase (JNK) inhibitors were designed and developed from a high-throughput-screening hit. Through the optimization of the piperazine amide 1, several potent compounds were discovered. The X-ray crystal structure of 4g showed a unique binding mode different from other well known JNK3 inhibitors.

Lysophosphatidylcholine up-regulates human endothelial nitric oxide synthase gene transactivity by c-Jun N-terminal kinase signalling pathway

PubMed Central

Xing, Feiyue; Liu, Jing; Mo, Yongyan; Liu, Zhifeng; Qin, Qinghe; Wang, Jingzhen; Fan, Zhenhua; Long, Yutian; Liu, Na; Zhao, Kesen; Jiang, Yong

2009-01-01

Human endothelial nitric oxide synthase (eNOS) plays a pivotal role in maintaining blood pressure homeostasis and vascular integrity. It has recently been reported that mitogen-activated protein kinases (MAPKs) are intimately implicated in expression of eNOS. However detailed mechanism mediated by them remains to be clarified. In this study, eNOS gene transactivity in human umbilical vein endothelial cells was up-regulated by stimulation of lysophosphatidylcholine (LPC). The stimulation of LPC highly activated both extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK), with differences in the dynamic processes of activation between them. Unexpectedly, p38 MAPK could not be activated by the stimulation of LPC. The activation of JNK signalling pathway by overexpression of JNK or its upstream kinase active mutant up-regulated the transactivity of eNOS significantly, but the activation of p38 signalling pathway down-regulated it largely. The inhibition of either ERK1/2 or JNK signalling pathway by kinase-selective inhibitors could markedly block the induction of the transactivity by LPC. It was observed by electrophoretic mobility shift assay that LPC stimulated both SP1 and AP1 DNA binding activity to go up. Additionally using decoy oligonucleotides proved that SP1 was necessary for maintaining the basal or stimulated transactivity, whereas AP1 contributed mainly to the increase of the stimulated transactivity. These findings indicate that the up-regulation of the eNOS gene transactivity by LPC involves the enhancement of SP1 transcription factor by the activation of JNK and ERK1/2 signalling pathways and AP1 transcription factor by the activation of JNK signalling pathway. PMID:18624763

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

PubMed

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

Inhibition of spinal astrocytic c-Jun N-terminal kinase (JNK) activation correlates with the analgesic effects of ketamine in neuropathic pain

PubMed Central

2011-01-01

Background We have previously reported that inhibition of astrocytic activation contributes to the analgesic effects of intrathecal ketamine on spinal nerve ligation (SNL)-induced neuropathic pain. However, the underlying mechanisms are still unclear. c-Jun N-terminal kinase (JNK), a member of mitogen-activated protein kinase (MAPK) family, has been reported to be critical for spinal astrocytic activation and neuropathic pain development after SNL. Ketamine can decrease lipopolysaccharide (LPS)-induced phosphorylated JNK (pJNK) expression and could thus exert its anti-inflammatory effect. We hypothesized that inhibition of astrocytic JNK activation might be involved in the suppressive effect of ketamine on SNL-induced spinal astrocytic activation. Methods Immunofluorescence histochemical staining was used to detect SNL-induced spinal pJNK expression and localization. The effects of ketamine on SNL-induced mechanical allodynia were confirmed by behavioral testing. Immunofluorescence histochemistry and Western blot were used to quantify the SNL-induced spinal pJNK expression after ketamine administration. Results The present study showed that SNL induced ipsilateral pJNK up-regulation in astrocytes but not microglia or neurons within the spinal dorsal horn. Intrathecal ketamine relieved SNL-induced mechanical allodynia without interfering with motor performance. Additionally, intrathecal administration of ketamine attenuated SNL-induced spinal astrocytic JNK activation in a dose-dependent manner, but not JNK protein expression. Conclusions The present results suggest that inhibition of JNK activation may be involved in the suppressive effects of ketamine on SNL-induced spinal astrocyte activation. Therefore, inhibition of spinal JNK activation may be involved in the analgesic effects of ketamine on SNL-induced neuropathic pain. PMID:21255465

[Oxidative stress promotes hepatocyte apoptosis mediated by glycogen synthase kinase 3β].

PubMed

Zhang, Xiangying; Guo, Yuanyuan; Zhang, Li; Wen, Tao; Piao, Zhengfu; Shi, Hongbo; Chen, Dexi; Duan, Zhongping; Ren, Feng

2015-01-01

To analyze the role of glycogen synthase kinase 3β (GSK3β) in hepatocyte apoptosis induced by oxidative stress. Human HL-7702 hepatoma cells were induced by H₂O₂/antimycin A to establish oxidative stress-induced cell apoptosis models. SB216763, a specific inhibitor of GSK3β, was given to the cells two hours before H₂O₂/antimycin A induction. Cell survival was observed using calcein acetoxymethyl ester/propidium iodide (PI) double staining, and cell apoptosis was detected using annexin V-FITC/PI staining combined with flow cytometry. In the meanwhile, the cell culture supernatant was subjected to lactate dehydrogenase (LDH) assay to evaluate the extent of cell death. The expressions of p-GSK3β, GSK3β, caspase-3, cleaved caspase-3, c-Jun N-terminal kinase (JNK) and cytochrome C (CytC) proteins were examined using Western blotting. Oxidative stress triggered by H₂O₂/antimycin A promoted GSK3β activity; inhibition of GSK3β activity by SB216763 relieved oxidative stress and reduced cell apoptosis induced by oxidative stress. Compared with the model groups, SB216763 intervened group showed that the cell apoptosis rate and the level of LDH were reduced significantly, and that the expressions of cleaved caspase-3, JNK, CytC proteins decreased. GSK3β is an important signaling molecule in the apoptosis pathway induced by oxidative stress. The inhibition on GSK3β may alleviate the oxidative stress-induced hepatocyte apoptosis.

Phosphorylation of Icariin Can Alleviate the Oxidative Stress Caused by the Duck Hepatitis Virus A through Mitogen-Activated Protein Kinases Signaling Pathways.

PubMed

Xiong, Wen; Zhang, Wei; Yuan, Wenjuan; Du, Hongxu; Ming, Ke; Yao, Fangke; Bai, Jingying; Chen, Yun; Liu, Jiaguo; Wang, Deyun; Hu, Yuanliang; Wu, Yi

2017-01-01

The duck virus hepatitis (DVH) caused by the duck hepatitis virus A (DHAV) has produced extensive economic losses to the duck industry. The currently licensed commercial vaccine has shown some defects and does not completely prevent the DVH. Accordingly, a new alternative treatment for this disease is urgently needed. Previous studies have shown that icariin (ICA) and its phosphorylated derivative (pICA) possessed good anti-DHAV effects through direct and indirect antiviral pathways, such as antioxidative stress. But the antioxidant activity showed some differences between ICA and pICA. The aim of this study is to prove that ICA and pICA attenuate oxidative stress caused by DHAV in vitro and in vivo , and to investigate their mechanism of action to explain their differences in antioxidant activities. In vivo , the dynamic deaths, oxidative evaluation indexes and hepatic pathological change scores were detected. When was added the hinokitiol which showed the pro-oxidative effect as an intervention method, pICA still possessed more treatment effect than ICA. The strong correlation between mortality and oxidative stress proves that ICA and pICA alleviate oxidative stress caused by DHAV. This was also demonstrated by the addition of hydrogen peroxide (H2O2) as an intervention method in vitro . pICA can be more effective than ICA to improve duck embryonic hepatocytes (DEHs) viability and reduce the virulence of DHAV. The strong correlation between TCID50 and oxidative stress demonstrates that ICA and pICA can achieve anti-DHAV effects by inhibiting oxidative stress. In addition, the superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) of ICA and pICA showed significant difference. pICA could significantly inhibit the phosphorylation of p38, extra cellular signal regulated Kinase (ERK 1/2) and c-Jun N-terminal kinase (JNK), which were related to mitogen-activated protein kinases (MAPKs) signaling pathways. Ultimately, compared to ICA, pICA exhibited more

[Effect of Acupuncture Intervention on c-jun N-terminal Kinase Signaling in the Hippocampus in Rats with Forced Swimming Stress].

PubMed

Guo, Yu; Xu, Ke; Bao, Wu-ye; Wang, Yu; Zhang, Xu-hui; Xu, Ming-min; Yu, Miao; Zhang, Chun-tao; Zhao, Bing-cong; Wu, Ji-hong; Tu, Ya

2016-02-01

To observe the effect of acupuncture on c-jun N-terminal Kinase (JNK) signaling in the hippocampus in rats with forced-swimming stress, so as to reveal its underlying mechanism in relieving depression-like motor response. Forty-eight Sprague-Dawley rats were randomly divided into 8 groups as control, control + JNK inhibitor (SP 600125) , model, model + SP 600125, acupuncture, acupuncture + SP 600125, Fluoxetine (an anti-depressant) , and Fluoxetine + SP 600125 (n = 6 in each group). The depression-like behavior (immobility) model was established by forcing the rat to swim in a glass-cylinder and solitary raise. Acupuncture stimulation was applied to "Baihui" (GV-20) and "Yintang" (GV 29) for 20 min before forced swimming and once again 24 h later.. The rats of the Fluoxetine and Fluoxetine+ SP 600125 groups were treated by intragastric administration of fluoxetine 10 mL (1.8 mg)/kg before forced swimming and once again 24 h thereafter. The rats of the model + SP 600125 and acupuncture + SP 600125 groups were treated by intraperitoneal injection of SP 600125 (10 mg/kg) 90 min before forced swimming and 30 min before acupuncture intervention, respectively. The immobility duration of rats in the water glass-cylinder was used to assess their depression-like behavior response. The expression levels of protein kinase kinase 4 (MKK 4), MKK 7, JNK, and phosphorylated JNK (p-JNK) in the hippocampus were detected by Western blot. Compared to the control group, the duration of immobility, and the expression levels of hippocampal MKK 4, MKK 7, and p-JNK proteins were significantly increased in the model group (P < 0.01). While in comparison with the model group, the duration of immobility in the model + SP 600125, acupuncture, acupuncture + SP 600125, Fluoxetine and Fluoxetine + SP 600125 groups, the expression levels of hippocampal MKK 4 and MKK 7 proteins in the Fluoxetine + SP 600125 group, and those of p-JNK protein in the acupuncture, acupuncture + SP 600125, model + SP

Mitomycin-C induces the apoptosis of human Tenon's capsule fibroblast by activation of c-Jun N-terminal kinase 1 and caspase-3 protease.

PubMed

Seong, Gong Je; Park, Channy; Kim, Chan Yoon; Hong, Young Jae; So, Hong-Seob; Kim, Sang-Duck; Park, Raekil

2005-10-01

To investigate whether mitochondrial dysfunction and mitogen-activated protein kinase family proteins are implicated in apoptotic signaling of human Tenon's capsule fibroblasts (HTCFs) by mitomycin-C. Apoptosis was determined by Hoechst nuclei staining, agarose gel electrophoresis, and flow cytometry in HTCFs treated with 0.4 mg/mL mitomycin-C for 5 minutes. Enzymatic digestion of florigenic biosubstrate assessed the catalytic activity of caspase proteases, including caspase-3, caspase-8, and caspase-9. Phosphotransferase activity of c-Jun N-terminal kinase (JNK) 1 was measured by in vitro immune complex kinase assay using c-Jun(1-79) protein as a substrate. Mitochondrial membrane potential transition (MPT) was measured by flow cytometric analysis of JC-1 staining. Mitomycin-C (0.4 mg/mL) induced the apoptosis of HTCFs, which was characterized as nucleic acid and genomic DNA fragmentation, chromatin condensation, and sub-G(0)/G(1) fraction of cell cycle increase. The catalytic activity of caspase-3 and caspase-9 was significantly increased and was accompanied by cytosolic release of cytochrome c and MPT in response to mitomycin-C. Treatment with mitomycin-C resulted in the increased expression of Fas, FasL, Bad, and phosphorylated p53 and a decreased level of phosphorylated AKT. Treatment with mitomycin-C also increased the phosphotransferase activity and tyrosine phosphorylation of JNK1, whose inhibitor significantly suppressed the cytotoxicity of mitomycin-C. Mitomycin-C induced the apoptosis of HTCFs through the activation of intrinsic and extrinsic caspase cascades with mitochondrial dysfunction. It also activated Fas-mediated apoptotic signaling of fibroblasts. Furthermore, the activation of JNK1 played a major role in the cytotoxicity of mitomycin-C.

Structure of the two-domain hexameric APS kinase from Thiobacillus denitrificans: structural basis for the absence of ATP sulfurylase activity

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

Gay, Sean C.; Segel, Irwin H.; Fisher, Andrew J., E-mail: fisher@chem.ucdavis.edu

2009-10-01

APS kinase from Thiobacillus denitrificans contains an inactive N-terminal ATP sulfurylase domain. The structure presented unveils the first hexameric assembly for an APS kinase, and reveals that structural changes in the N-terminal domain disrupt the ATP sulfurylase active site thus prohibiting activity. The Tbd-0210 gene of the chemolithotrophic bacterium Thiobacillus denitrificans is annotated to encode a 60.5 kDa bifunctional enzyme with ATP sulfurylase and APS kinase activity. This putative bifunctional enzyme was cloned, expressed and structurally characterized. The 2.95 Å resolution X-ray crystal structure reported here revealed a hexameric assembly with D{sub 3} symmetry. Each subunit contains a large N-terminalmore » sulfurylase-like domain and a C-terminal APS kinase domain reminiscent of the two-domain fungal ATP sulfurylases of Penicillium chrysogenum and Saccharomyces cerevisiae, which also exhibit a hexameric assembly. However, the T. denitrificans enzyme exhibits numerous structural and sequence differences in the N-terminal domain that render it inactive with respect to ATP sulfurylase activity. Surprisingly, the C-terminal domain does indeed display APS kinase activity, indicating that this gene product is a true APS kinase. Therefore, these results provide the first structural insights into a unique hexameric APS kinase that contains a nonfunctional ATP sulfurylase-like domain of unknown function.« less

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

PubMed

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

1997-02-15

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

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

PubMed

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.

KFC, a Ste20-like kinase with mitogenic potential and capability to activate the SAPK/JNK pathway.

PubMed

Yustein, J T; Li, D; Robinson, D; Kung, H J

2000-02-03

The Sterile-20 (Ste20) family of serine-threonine kinases has been implicated in the activation of the stress-activated protein kinase pathways. However, the physiological role has remained ambiguous for most of the investigated mammalian Ste20's. Here we report the cloning of a novel Ste20-like kinase, from chicken embryo fibroblast (CEF) cells, which we have named KFC, for Kinase From Chicken. The 898 amino acid full-length KFC protein contains an amino-terminal kinase domain, an adjacent downstream serine-rich region, and a C-terminal tail containing a coiled-coil domain. Here we show that the coiled-coil domain of KFC negatively regulates the intrinsic kinase activity. We have also identified a splice variant of KFC in which there is a 207 nucleotide in-frame deletion. This deletion of 69 amino acids encompasses the serine-rich region. These two isoforms, called KFCL, for full-length, and KFCS for spliced (or short) form, not only differ in structure, but also in biological properties. Stable CEF cells overexpressing KFCL, but not KFCS, have a significant increase in growth rate when compared to parental cells. This mitogenic effect is the first such reported for this family of kinases. Finally, we found that KFC, when activated by truncation of the regulatory C-terminus, has a specific activation of the stress-activated protein kinase (SAPK/JNK) pathway.

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5'-Terminal Cap and Hairpin.

PubMed

Korneeva, Nadejda L; Song, Anren; Gram, Hermann; Edens, Mary Ann; Rhoads, Robert E

2016-02-12

The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Theoretical Insights Reveal Novel Motions in Csk’s SH3 Domain That Control Kinase Activation

PubMed Central

Barkho, Sulyman; Pierce, Levi C. T.; Li, Sheng; Adams, Joseph A.; Jennings, Patricia A.

2015-01-01

The Src family of tyrosine kinases (SFKs) regulate numerous aspects of cell growth and differentiation and are under the principal control of the C-terminal Src Kinase (Csk). Although Csk and SFKs share conserved kinase, SH2 and SH3 domains, they differ considerably in three-dimensional structure, regulatory mechanism, and the intrinsic kinase activities. Although the SH2 and SH3 domains are known to up- or down-regulate tyrosine kinase function, little is known about the global motions in the full-length kinase that govern these catalytic variations. We use a combination of accelerated Molecular Dynamics (aMD) simulations and experimental methods to provide a new view of functional motions in the Csk scaffold. These computational studies suggest that high frequency vibrations in the SH2 domain are coupled through the N-terminal lobe of the kinase domain to motions in the SH3 domain. The effects of these reflexive movements on the kinase domain can be viewed using both Deuterium Exchange Mass Spectrometry (DXMS) and steady-state kinetic methods. Removal of several contacts, including a crystallographically unobserved N-terminal segment, between the SH3 and kinase domains short-circuit these coupled motions leading to reduced catalytic efficiency and stability of N-lobe motifs within the kinase domain. The data expands the model of Csk’s activation whereby separate domains productively interact with two diametrically opposed surfaces of the kinase domain. Such reversible transitions may organize the active structure of the tyrosine kinase domain of Csk. PMID:26030592

β-subunit myristoylation functions as an energy sensor by modulating the dynamics of AMP-activated Protein Kinase.

PubMed

Ali, Nada; Ling, Naomi; Krishnamurthy, Srinath; Oakhill, Jonathan S; Scott, John W; Stapleton, David I; Kemp, Bruce E; Anand, Ganesh Srinivasan; Gooley, Paul R

2016-12-21

The heterotrimeric AMP-activated protein kinase (AMPK), consisting of α, β and γ subunits, is a stress-sensing enzyme that is activated by phosphorylation of its activation loop in response to increases in cellular AMP. N-terminal myristoylation of the β-subunit has been shown to suppress Thr172 phosphorylation, keeping AMPK in an inactive state. Here we use amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the structural and dynamic properties of the mammalian myristoylated and non-myristoylated inactivated AMPK (D139A) in the presence and absence of nucleotides. HDX MS data suggests that the myristoyl group binds near the first helix of the C-terminal lobe of the kinase domain similar to other kinases. Our data, however, also shows that ATP.Mg 2+ results in a global stabilization of myristoylated, but not non-myristoylated AMPK, and most notably for peptides of the activation loop of the α-kinase domain, the autoinhibitory sequence (AIS) and the βCBM. AMP does not have that effect and HDX measurements for myristoylated and non-myristoylated AMPK in the presence of AMP are similar. These differences in dynamics may account for a reduced basal rate of phosphorylation of Thr172 in myristoylated AMPK in skeletal muscle where endogenous ATP concentrations are very high.

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

PubMed Central

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

1997-01-01

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

A peptide representing the carboxyl-terminal tail of the met receptor inhibits kinase activity and invasive growth.

PubMed

Bardelli, A; Longati, P; Williams, T A; Benvenuti, S; Comoglio, P M

1999-10-08

Interaction of the hepatocyte growth factor (HGF) with its receptor, the Met tyrosine kinase, results in invasive growth, a genetic program essential to embryonic development and implicated in tumor metastasis. Met-mediated invasive growth requires autophosphorylation of the receptor on tyrosines located in the kinase activation loop (Tyr(1234)-Tyr(1235)) and in the carboxyl-terminal tail (Tyr(1349)-Tyr(1356)). We report that peptides derived from the Met receptor tail, but not from the activation loop, bind the receptor and inhibit the kinase activity in vitro. Cell delivery of the tail receptor peptide impairs HGF-dependent Met phosphorylation and downstream signaling. In normal and transformed epithelial cells, the tail receptor peptide inhibits HGF-mediated invasive growth, as measured by cell migration, invasiveness, and branched morphogenesis. The Met tail peptide inhibits the closely related Ron receptor but does not significantly affect the epidermal growth factor, platelet-derived growth factor, or vascular endothelial growth factor receptor activities. These experiments show that carboxyl-terminal sequences impair the catalytic properties of the Met receptor, thus suggesting that in the resting state the nonphosphorylated tail acts as an intramolecular modulator. Furthermore, they provide a strategy to selectively target the MET proto-oncogene by using small, cell-permeable, peptide derivatives.

Mitogen- and Stress-Activated Kinase 1 (MSK1) Regulates Cigarette Smoke-Induced Histone Modifications on NF-κB-dependent Genes

PubMed Central

Sundar, Isaac K.; Chung, Sangwoon; Hwang, Jae-woong; Lapek, John D.; Bulger, Michael; Friedman, Alan E.; Yao, Hongwei; Davie, James R.; Rahman, Irfan

2012-01-01

Cigarette smoke (CS) causes sustained lung inflammation, which is an important event in the pathogenesis of chronic obstructive pulmonary disease (COPD). We have previously reported that IKKα (I kappaB kinase alpha) plays a key role in CS-induced pro-inflammatory gene transcription by chromatin modifications; however, the underlying role of downstream signaling kinase is not known. Mitogen- and stress-activated kinase 1 (MSK1) serves as a specific downstream NF-κB RelA/p65 kinase, mediating transcriptional activation of NF-κB-dependent pro-inflammatory genes. The role of MSK1 in nuclear signaling and chromatin modifications is not known, particularly in response to environmental stimuli. We hypothesized that MSK1 regulates chromatin modifications of pro-inflammatory gene promoters in response to CS. Here, we report that CS extract activates MSK1 in human lung epithelial (H292 and BEAS-2B) cell lines, human primary small airway epithelial cells (SAEC), and in mouse lung, resulting in phosphorylation of nuclear MSK1 (Thr581), phospho-acetylation of RelA/p65 at Ser276 and Lys310 respectively. This event was associated with phospho-acetylation of histone H3 (Ser10/Lys9) and acetylation of histone H4 (Lys12). MSK1 N- and C-terminal kinase-dead mutants, MSK1 siRNA-mediated knock-down in transiently transfected H292 cells, and MSK1 stable knock-down mouse embryonic fibroblasts significantly reduced CS extract-induced MSK1, NF-κB RelA/p65 activation, and posttranslational modifications of histones. CS extract/CS promotes the direct interaction of MSK1 with RelA/p65 and p300 in epithelial cells and in mouse lung. Furthermore, CS-mediated recruitment of MSK1 and its substrates to the promoters of NF-κB-dependent pro-inflammatory genes leads to transcriptional activation, as determined by chromatin immunoprecipitation. Thus, MSK1 is an important downstream kinase involved in CS-induced NF-κB activation and chromatin modifications, which have implications in pathogenesis

N-acetylcysteine protects against cadmium-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in testes.

PubMed

Ji, Yan-Li; Wang, Hua; Zhang, Cheng; Zhang, Ying; Zhao, Mei; Chen, Yuan-Hua; Xu, De-Xiang

2013-03-01

Cadmium (Cd) is a reproductive toxicant that induces germ cell apoptosis in the testes. Previous studies have demonstrated that endoplasmic reticulum (ER) stress is involved in Cd-induced germ cell apoptosis. The aim of the present study was to investigate the effects of N-acetylcysteine (NAC), an antioxidant, on Cd-induced ER stress and germ cell apoptosis in the testes. Male CD-1 mice were intraperitoneally injected with CdCl2 (2.0 mg kg(-1)). As expected, acute Cd exposure induced germ cell apoptosis in the testes, as determined by terminal dUTP nick-end labelling (TUNEL). However, the administration of NAC alleviated Cd-induced germ cell apoptosis in the testes. Further analysis showed that NAC attenuated the Cd-induced upregulation of testicular glucose-regulated protein 78 (GRP78), an important ER molecular chaperone. Moreover, NAC inhibited the Cd-induced phosphorylation of testicular eukaryotic translation initiation factor 2α (eIF2α), a downstream target of the double-stranded RNA-activated kinase-like ER kinase (PERK) pathway. In addition, NAC blocked the Cd-induced activation of testicular X binding protein (XBP)-1, indicating that NAC attenuates the Cd-induced ER stress and the unfolded protein response (UPR). Interestingly, NAC almost completely prevented the Cd-induced elevation of C/EBP homologous protein (CHOP) and phosphorylation of c-Jun N-terminal kinase (JNK), two components of the ER stress-mediated apoptotic pathway. In conclusion, NAC protects against Cd-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in the testes.

N-acetylcysteine protects against cadmium-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in testes

PubMed Central

Ji, Yan-Li; Wang, Hua; Zhang, Cheng; Zhang, Ying; Zhao, Mei; Chen, Yuan-Hua; Xu, De-Xiang

2013-01-01

Cadmium (Cd) is a reproductive toxicant that induces germ cell apoptosis in the testes. Previous studies have demonstrated that endoplasmic reticulum (ER) stress is involved in Cd-induced germ cell apoptosis. The aim of the present study was to investigate the effects of N-acetylcysteine (NAC), an antioxidant, on Cd-induced ER stress and germ cell apoptosis in the testes. Male CD-1 mice were intraperitoneally injected with CdCl2 (2.0 mg kg−1). As expected, acute Cd exposure induced germ cell apoptosis in the testes, as determined by terminal dUTP nick-end labelling (TUNEL). However, the administration of NAC alleviated Cd-induced germ cell apoptosis in the testes. Further analysis showed that NAC attenuated the Cd-induced upregulation of testicular glucose-regulated protein 78 (GRP78), an important ER molecular chaperone. Moreover, NAC inhibited the Cd-induced phosphorylation of testicular eukaryotic translation initiation factor 2α (eIF2α), a downstream target of the double-stranded RNA-activated kinase-like ER kinase (PERK) pathway. In addition, NAC blocked the Cd-induced activation of testicular X binding protein (XBP)-1, indicating that NAC attenuates the Cd-induced ER stress and the unfolded protein response (UPR). Interestingly, NAC almost completely prevented the Cd-induced elevation of C/EBP homologous protein (CHOP) and phosphorylation of c-Jun N-terminal kinase (JNK), two components of the ER stress-mediated apoptotic pathway. In conclusion, NAC protects against Cd-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in the testes. PMID:23353715

[On the mechanism of noopept action: decrease in activity of stress-induced kinases and increase in expression of neutrophines].

PubMed

Ostrovskaia, R U; Vakhitova, Iu V; Salimgareeva, M Kh; Iamidanov, R S; Sadovnikov, S V; Kapitsa, I G; Seredenin, S B

2010-12-01

The influence of noopept (N-phenylacetyl-L-prolylglycine ethyl ester, GVS-111)--a drug combining the nootrope and neuroprotector properties--on the activity of mitogen-activated protein kinases (MAPKs) and the level of NGF and BDNF gene and protein expression in the frontal cortex, hippocampus, and hypothalamus has been studied in rats. Under conditions of chronic administration (28 days, 0.5 mg/day, i.p.), noopept decreased the activity of stress-induced kinases (SAPK/JNK 46/54 and pERK1/2) in rat hippocampus and increases the level of mRNA of the BDNF gene in both hypothalamus and hippocampus. The content of BDNF protein in the hypothalamus was also somewhat increased. In the context of notions about the activation of stress-induced kinases, as an important factor of amyloidogenesis and tau-protein deposition in brain tissue, and the role of deficiency of the neurotrophic factors in the development of neurodegenerative processes, the observed decrease in the activity of stress-activated MAPKs and increased expression of BDNF as a result of noopept administration suggest thatthis drug hasaspecific activity withrespect to some pathogenetic mechanisms involved in the Alzheimer disease.

Daphnoretin modulates differentiation and maturation of human dendritic cells through down-regulation of c-Jun N-terminal kinase.

PubMed

Chen, Chien-An; Liu, Chien-Kuo; Hsu, Ming-Ling; Chi, Chih-Wen; Ko, Chun-Chuan; Chen, Jian-Syun; Lai, Cheng-Ta; Chang, Hen-Hong; Lee, Tzung-Yan; Lai, Yuen-Liang; Chen, Yu-Jen

2017-10-01

Daphnoretin, an active constituent of Wikstroemia indica C.A. Meys, has been shown possessing anti-cancer activity. In this study, we examined the effect of daphnoretin on differentiation and maturation of human myeloid dendritic cells (DCs). After treatment with daphnoretin (0, 1.1, 3.3, 10 and 30μM) to initiate monocytes, the recovery rate of DCs was reduced in a dose-dependent manner. The mature DCs differentiated in the presence of daphnoretin had fewer and shorter dendrites. Daphnoretin modulated DCs differentiation and maturation in terms of lower expression of CD1a, CD40, CD83, DC-SIGN, and HLA-DR. Daphnoretin inhibited the allostimulatory activity of DCs on proliferation of naive CD4 + CD45 + RA + T cell. On the mitogen-activated protein kinase, daphnoretin down-regulated the lipopolysaccharide-augmented expression of phosphorylated c-Jun N-terminal kinase (pJNK), but not p38 and extracellular signal-regulated kinase 1/2 (ERK1/2). Activation of JNK by anisomycin reversed the effect of daphnoretin on daphnoretin-inhibited pJNK expression and dendrite formation of DCs. In disease model related to maturation of DCs, daphnoretin suppressed the acute rejection of skin allografts in mice. Our results suggest that daphnoretin modulated differentiation and maturation of DCs toward a state of atypical maturation with impaired allostimulatory function and this effect may go through down-regulation of phosphorylated JNK. Copyright © 2017 Elsevier B.V. All rights reserved.

Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases

PubMed Central

Cargnello, Marie; Roux, Philippe P.

2011-01-01

Summary: The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (α, β, γ, and δ), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries. PMID:21372320

Inhibition of Vascular c-Jun N-Terminal Kinase 2 Improves Obesity-Induced Endothelial Dysfunction After Roux-en-Y Gastric Bypass.

PubMed

Doytcheva, Petia; Bächler, Thomas; Tarasco, Erika; Marzolla, Vincenzo; Engeli, Michael; Pellegrini, Giovanni; Stivala, Simona; Rohrer, Lucia; Tona, Francesco; Camici, Giovanni G; Vanhoutte, Paul M; Matter, Christian M; Lutz, Thomas A; Lüscher, Thomas F; Osto, Elena

2017-11-14

Roux-en-Y gastric bypass (RYGB) reduces obesity-associated comorbidities and cardiovascular mortality. RYGB improves endothelial dysfunction, reducing c-Jun N-terminal kinase (JNK) vascular phosphorylation. JNK activation links obesity with insulin resistance and endothelial dysfunction. Herein, we examined whether JNK1 or JNK2 mediates obesity-induced endothelial dysfunction and if pharmacological JNK inhibition can mimic RYGB vascular benefits. After 7 weeks of a high-fat high-cholesterol diet, obese rats underwent RYGB or sham surgery; sham-operated ad libitum-fed rats received, for 8 days, either the control peptide D-TAT or the JNK peptide inhibitor D-JNKi-1 (20 mg/kg per day subcutaneous). JNK peptide inhibitor D-JNKi-1 treatment improved endothelial vasorelaxation in response to insulin and glucagon-like peptide-1, as observed after RYGB. Obesity increased aortic phosphorylation of JNK2, but not of JNK1. RYGB and JNK peptide inhibitor D-JNKi-1 treatment blunted aortic JNK2 phosphorylation via activation of glucagon-like peptide-1-mediated signaling. The inhibitory phosphorylation of insulin receptor substrate-1 was reduced, whereas the protein kinase B/endothelial NO synthase pathway was increased and oxidative stress was decreased, resulting in improved vascular NO bioavailability. Decreased aortic JNK2 phosphorylation after RYGB rapidly improves obesity-induced endothelial dysfunction. Pharmacological JNK inhibition mimics the endothelial protective effects of RYGB. These findings highlight the therapeutic potential of novel strategies targeting vascular JNK2 against the severe cardiovascular disease associated with obesity. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

A Novel c-Jun N-terminal Kinase (JNK) Signaling Complex Involved in Neuronal Migration during Brain Development.

PubMed

Zhang, Feng; Yu, Jingwen; Yang, Tao; Xu, Dan; Chi, Zhixia; Xia, Yanheng; Xu, Zhiheng

2016-05-27

Disturbance of neuronal migration may cause various neurological disorders. Both the transforming growth factor-β (TGF-β) signaling and microcephaly-associated protein WDR62 are important for neuronal migration during brain development; however, the underlying molecular mechanisms involved remain unclear. We show here that knock-out or knockdown of Tak1 (TGFβ-activated kinase 1) and Jnk2 (c-Jun N-terminal kinase 2) perturbs neuronal migration during cortical development and that the migration defects incurred by knock-out and/or knockdown of Tβr2 (type II TGF-β receptor) or Tak1 can be partially rescued by expression of TAK1 and JNK2, respectively. Furthermore, TAK1 forms a protein complex with RAC1 and two scaffold proteins of the JNK pathway, the microcephaly-associated protein WDR62 and the RAC1-interacting protein POSH (plenty of Src homology). Components of the complex coordinate with each other in the regulation of TAK1 as well as JNK activities. We suggest that unique JNK protein complexes are involved in the diversified biological and pathological functions during brain development and pathogenesis of diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Involvement of protein kinase B and mitogen-activated protein kinases in experimental normothermic liver ischaemia-reperfusion injury.

PubMed

Cursio, R; Filippa, N; Miele, C; Van Obberghen, E; Gugenheim, J

2006-06-01

This study evaluated the role of protein kinase B (PKB), phosphatidylinositol 3-kinase (PI3-K), Bcl-2-associated death protein (BAD) and mitogen-activated protein kinases (MAPKs) in normothermic ischaemia-reperfusion (IR)-induced apoptosis in rat liver. Rats were divided into two groups that received either phosphate-buffered saline (control) or the caspase inhibitor Z-Asp-2,6-dichorobenzoyloxymethylketone (Z-Asp-cmk), injected intravenously 2 min before the induction of 120 min of normothermic liver ischaemia. Liver apoptosis was assessed by the terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) method. PI3-K, PKB, BAD and MAPK activities were measured in ischaemic and non-ischaemic lobes at various times after reperfusion. The number of TUNEL-positive cells was significantly decreased after pretreatment with Z-Asp-cmk. In controls, PI3-K and PKB activities and BAD phosphorylation were inhibited in ischaemic liver lobes. The MAPKs (extracellular signal-regulated kinases, c-Jun N-terminal kinase and p38) showed different patterns of activation during IR. PKB activity was not modified by pretreatment with Z-Asp-cmk. Induction of apoptosis during IR liver injury might be triggered by inactivation of the antiapoptotic PI3-K-PKB pathway and activation of the proapoptotic MAPKs. Copyright (c) 2006 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

Mapping of Functional Domains of the Lipid Kinase Phosphatidylinositol 4-Kinase Type III Alpha Involved in Enzymatic Activity and Hepatitis C Virus Replication

PubMed Central

Harak, Christian; Radujkovic, Danijela; Taveneau, Cyntia; Reiss, Simon; Klein, Rahel; Bressanelli, Stéphane

2014-01-01

ABSTRACT The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an endoplasmic reticulum (ER)-resident enzyme that synthesizes phosphatidylinositol 4-phosphate (PI4P). PI4KIIIα is an essential host factor for hepatitis C virus (HCV) replication. Interaction with HCV nonstructural protein 5A (NS5A) leads to kinase activation and accumulation of PI4P at intracellular membranes. In this study, we investigated the structural requirements of PI4KIIIα in HCV replication and enzymatic activity. Therefore, we analyzed PI4KIIIα mutants for subcellular localization, reconstitution of HCV replication in PI4KIIIα knockdown cell lines, PI4P induction in HCV-positive cells, and lipid kinase activity in vitro. All mutants still interacted with NS5A and localized in a manner similar to that of the full-length enzyme, suggesting multiple regions of PI4KIIIα are involved in NS5A interaction and subcellular localization. Interestingly, the N-terminal 1,152 amino acids were dispensable for HCV replication, PI4P induction, and enzymatic function, whereas further N-terminal or C-terminal deletions were deleterious, thereby defining the minimal PI4KIIIα core enzyme at a size of ca. 108 kDa. Additional deletion of predicted functional motifs within the C-terminal half of PI4KIIIα also were detrimental for enzymatic activity and for the ability of PI4KIIIα to rescue HCV replication, with the exception of a proposed nuclear localization signal, suggesting that the entire C-terminal half of PI4KIIIα is involved in the formation of a minimal enzymatic core. This view was supported by structural modeling of the PI4KIIIα C terminus, suggesting a catalytic center formed by an N- and C-terminal lobe and an armadillo-fold motif, which is preceded by three distinct alpha-helical domains probably involved in regulation of enzymatic activity. IMPORTANCE The lipid kinase PI4KIIIα is of central importance for cellular phosphatidylinositol metabolism and is a key host cell

Induction of filopodia-like protrusions in N1E-115 neuroblastoma cells by diacylglycerol kinase γ independent of its enzymatic activity: potential novel function of the C-terminal region containing the catalytic domain of diacylglycerol kinase γ.

PubMed

Tanino, Fumihiko; Maeda, Yuki; Sakai, Hiromichi; Sakane, Fumio

2013-01-01

Type I diacylglycerol kinase (DGK) isozymes (α, β, and γ) contain recoverin homology domains and calcium-binding EF-hand motifs at their N-termini. The γ-isoform of DGK is abundantly expressed in retinal and Purkinje cells; however, its function in neuronal cells remains unknown. Here, we report that the mRNA and protein levels of DGKγ, but not DGKα or β, were markedly increased in N1E-115 neuroblastoma cells upon cellular differentiation by serum starvation. Interestingly, overexpression of wild-type DGKγ, which was partially located at the plasma membrane, considerably induced the formation of slender, filopodia-like cytoplasmic projections from N1E-115 cell bodies. Deletion of the recoverin homology domain and the EF-hand motifs, which potentiated the plasma membrane localization of the isozyme, significantly enhanced the formation of the filopodia-like protrusions. Intriguingly, the catalytic activity of the isozyme is not essential for the protrusion formation. The N-terminal half of the catalytic domain and a short stretch of amino acid residues at the C-terminus are responsible for plasma membrane localization and filopodia-like process formation. Taken together, we have described a potentially novel morphological function of the C-terminal DGKγ catalytic region that is independent of its enzymatic activity.

The molecular architecture of human N-acetylgalactosamine kinase.

PubMed

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.

Oncoprotein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning

2001-02-27

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

Nuclear translocation of doublecortin-like protein kinase and phosphorylation of a transcription factor JDP2

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

Nagamine, Tadashi; Nomada, Shohgo; Onouchi, Takashi

2014-03-28

Highlights: • Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase. • In living cells, DCLK was cleaved into two functional fragments. • zDCLK(kinase) was translocated into the nucleus by osmotic stresses. • Jun dimerization protein 2 (JDP2) was identified as zDCLK(kinase)-binding protein. • JDP2 was efficiently phosphorylated by zDCLK(kinase) only when histone was present. - Abstract: Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase predominantly expressed in brain. In a previous paper, we reported that zebrafish DCLK2 (zDCLK) was cleaved into two functional fragments; the N-terminal zDCLK(DC + SP) with microtubule-binding activity and the C-terminal zDCLK(kinase) with amore » Ser/Thr protein kinase activity. In this study, we demonstrated that zDCLK(kinase) was widely distributed in the cytoplasm and translocated into the nucleus when the cells were treated under hyperosmotic conditions with NaCl or mannitol. By two-hybrid screening using the C-terminal domain of DCLK, Jun dimerization protein 2 (JDP2), a nuclear transcription factor, was identified as zDCLK(kinase)-binding protein. Furthermore, JDP2 served as an efficient substrate for zDCLK(kinase) only when histone was present. These results suggest that the kinase fragment of DCLK is translocated into the nucleus upon hyperosmotic stresses and that the kinase efficiently phosphorylates JDP2, a possible target in the nucleus, with the aid of histones.« less

Spm1, a stress-activated MAP kinase that regulates morphogenesis in S.pombe.

PubMed Central

Zaitsevskaya-Carter, T; Cooper, J A

1997-01-01

A gene encoding a novel MAP kinase family member, Spm1, was isolated from the fission yeast Schizosaccharomyces pombe. Overproduction of Spm1 inhibits proliferation. Disruption of the spm1+ gene interferes with cell separation and morphogenesis. Under conditions of nutrient limitation, hypertonic stress or elevated temperature, spm1 delta cells grow as short branched filaments in which the cell walls and septa are thickened, suggesting defects in polarized growth and cell wall remodeling. At high osmolarity, spm1 delta cells fail to form colonies. The Spm1 protein is tyrosine phosphorylated and activated in response to osmotic and heat stress, consistent with a role for Spm1 in adaptation to these conditions. Two other S.pombe MAP kinases are known, Spk1, required for sexual differentiation and sporulation, and Spc1/Sty1/Phh1, which is activated in hypertonic conditions. However, the distinctive features of the spm1 delta mutant phenotype and direct biochemical assays suggest that Spm1 does not lie on other known MAP kinase pathways. Our results demonstrate the existence of a new MAP kinase pathway that regulates cell wall remodeling and cytokinesis in response to environmental stresses. PMID:9135147

Saw palmetto extract suppresses insulin-like growth factor-I signaling and induces stress-activated protein kinase/c-Jun N-terminal kinase phosphorylation in human prostate epithelial cells.

PubMed

Wadsworth, Teri L; Carroll, Julie M; Mallinson, Rebecca A; Roberts, Charles T; Roselli, Charles E

2004-07-01

A common alternative therapy for benign prostatic hyperplasia (BPH) is the extract from the fruit of saw palmetto (SPE). BPH is caused by nonmalignant growth of epithelial and stromal elements of the prostate. IGF action is important for prostate growth and development, and changes in the IGF system have been documented in BPH tissues. The main signaling pathways activated by the binding of IGF-I to the IGF-I receptor (IGF-IR) are the ERK arm of the MAPK cascade and the phosphoinositol-3-kinase (PI3K)/protein kinase B (PKB/Akt) cascade. We tested the hypothesis that SPE suppresses growth and induces apoptosis in the P69 prostate epithelial cell line by inhibiting IGF-I signaling. Treatment with 150 microg/ml SPE for 24 h decreased IGF-I-induced proliferation of P69 cells and induced cleavage of the enzyme poly(ADP-ribose)polymerase (PARP), an index of apoptosis. Treatment of serum-starved P69 cells with 150 microg/ml SPE for 6 h reduced IGF-I-induced phosphorylation of Akt (assessed by Western blot) and Akt activity (assessed by an Akt kinase assay). Western blot analysis showed that SPE reduced IGF-I-induced phosphorylation of the adapter protein insulin receptor substrate-1 and decreased downstream effects of Akt activation, including increased cyclin D1 levels and phosphorylation of glycogen synthase kinase-3 and p70(s6k). There was no effect on IGF-I-induced phosphorylation of MAPK, IGF-IR, or Shc. Treatment of starved cells with SPE alone induced phosphorylation the proapoptotic protein JNK. SPE treatment may relieve symptoms of BPH, in part, by inhibiting specific components of the IGF-I signaling pathway and inducing JNK activation, thus mediating antiproliferative and proapoptotic effects on prostate epithelia.

Mitogen-activated protein kinase kinase 5 (MKK5)-mediated signalling cascade regulates expression of iron superoxide dismutase gene in Arabidopsis under salinity stress

PubMed Central

Xing, Yu; Chen, Wei-hua; Jia, Wensuo; Zhang, Jianhua

2015-01-01

Superoxide dismutases (SODs) are involved in plant adaptive responses to biotic and abiotic stresses but the upstream signalling process that modulates their expression is not clear. Expression of two iron SODs, FSD2 and FSD3, was significantly increased in Arabidopsis in response to NaCl treatment but blocked in transgenic MKK5-RNAi plant, mkk5. Using an assay system for transient expression in protoplasts, it was found that mitogen-activated protein kinase kinase 5 (MKK5) was also activated in response to salt stress. Overexpression of MKK5 in wild-type plants enhanced their tolerance to salt treatments, while mkk5 mutant exhibited hypersensitivity to salt stress in germination on salt-containing media. Moreover, another kinase, MPK6, was also involved in the MKK5-mediated iron superoxide dismutase (FSD) signalling pathway in salt stress. The kinase activity of MPK6 was totally turned off in mkk5, whereas the activity of MPK3 was only partially blocked. MKK5 interacted with the MEKK1 protein that was also involved in the salt-induced FSD signalling pathway. These data suggest that salt-induced FSD2 and FSD3 expressions are influenced by MEKK1 via MKK5–MPK6-coupled signalling. This MAP kinase cascade (MEKK1, MKK5, and MPK6) mediates the salt-induced expression of iron superoxide dismutases. PMID:26136265

Oncoprotein protein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning

1997-01-01

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

Oncoprotein protein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

2003-02-04

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

Oncoprotein protein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning

1997-01-01

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

Oncoprotein protein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning

1998-01-01

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

c-Jun N-terminal kinase 1 promotes transforming growth factor-β1-induced epithelial-to-mesenchymal transition via control of linker phosphorylation and transcriptional activity of Smad3.

PubMed

Velden, Jos L J van der; Alcorn, John F; Guala, Amy S; Badura, Elsbeth C H L; Janssen-Heininger, Yvonne M W

2011-04-01

Transforming growth factor (TGF)-β1 is a key mediator of lung remodeling and fibrosis. Epithelial cells are both a source of and can respond to TGF-β1 with epithelial-to-mesenchymal transition (EMT). We recently determined that TGF-β1-induced EMT in lung epithelial cells requires the presence of c-Jun N-terminal kinase (JNK) 1. Because TGF-β1 signals via Smad complexes, the goal of the present study was to determine the impact of JNK1 on phosphorylation of Smad3 and Smad3-dependent transcriptional responses in lung epithelial cells. Evaluation of JNK1-deficient lung epithelial cells demonstrated that TGF-β1-induced terminal phosphorylation of Smad3 was similar, whereas phosphorylation of mitogen-activated protein kinase sites in the linker regions of Smad3 was diminished, in JNK1-deficient cells compared with wild-type cells. In comparison to wild-type Smad3, expression of a mutant Smad3 in which linker mitogen-activated protein kinase sites were ablated caused a marked attenuation in JNK1 or TGF-β1-induced Smad-binding element transcriptional activity, and expression of plasminogen activator inhibitor-1, fibronectin-1, high-mobility group A2, CArG box-binding factor-A, and fibroblast-specific protein-1, genes critical in the process of EMT. JNK1 enhanced the interaction between Smad3 and Smad4, which depended on linker phosphorylation of Smad3. Conversely, Smad3 with phosphomimetic mutations in the linker domain further enhanced EMT-related genes and proteins, even in the absence of JNK1. Finally, we demonstrated a TGF-β1-induced interaction between Smad3 and JNK1. Collectively, these results demonstrate that Smad3 phosphorylation in the linker region and Smad transcriptional activity are directly or indirectly controlled by JNK1, and provide a putative mechanism whereby JNK1 promotes TGF-β1-induced EMT.

SH2 domains: modulators of nonreceptor tyrosine kinase activity.

PubMed

Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan

2009-12-01

The Src homology 2 (SH2) domain is a sequence-specific phosphotyrosine-binding module present in many signaling molecules. In cytoplasmic tyrosine kinases, the SH2 domain is located N-terminally to the catalytic kinase domain (SH1) where it mediates cellular localization, substrate recruitment, and regulation of kinase activity. Initially, structural studies established a role of the SH2 domain stabilizing the inactive state of Src family members. However, biochemical characterization showed that the presence of the SH2 domain is frequently required for catalytic activity, suggesting a crucial function stabilizing the active state of many nonreceptor tyrosine kinases. Recently, the structure of the SH2-kinase domain of Fes revealed that the SH2 domain stabilizes the active kinase conformation by direct interactions with the regulatory helix alphaC. Stabilizing interactions between the SH2 and the kinase domains have also been observed in the structures of active Csk and Abl. Interestingly, mutations in the SH2 domain found in human disease can be explained by SH2 domain destabilization or incorrect positioning of the SH2. Here we summarize our understanding of mechanisms that lead to tyrosine kinase activation by direct interactions mediated by the SH2 domain and discuss how mutations in the SH2 domain trigger kinase inactivation.

Effects of overexpression of IL-1 receptor-associated kinase on NFkappaB activation, IL-2 production and stress-activated protein kinases in the murine T cell line EL4.

PubMed

Knop, J; Wesche, H; Lang, D; Martin, M U

1998-10-01

The association and activation of the IL-1 receptor-associated protein kinase (IRAK) to the IL-1 receptor complex is one of the earliest events detectable in IL-1 signal transduction. We generated permanent clones of the murine T cell line EL4 6.1 overexpressing human (h)IRAK to evaluate the role of this kinase in IL-1 signaling. Overexpression of hIRAK enhanced IL-1-stimulated activation of the transcription factor NFkappaB, whereas a truncated form (N-IRAK) specifically inhibited IL-1-dependent NFkappaB activity. In clones stably overexpressing hIRAK a weak constitutive activation of NFkappaB correlated with a low basal IL-2 production which was enhanced in an IL-1-dependent manner. Compared to the parental cell line the dose-response curve of IL-1-induced IL-2 production was shifted in both potency and efficacy. These results demonstrate that IRAK directly triggers NFkappaB-mediated gene expression in EL4 cells. Qualitatively different effects were observed for the IL-1-induced activation of stress-activated protein (SAP) kinases: permanent overexpression of IRAK did not affect the dose dependence but prolonged the kinetics of IL-1-induced activation of SAP kinases, suggesting that this signaling branch may be regulated by distinct mechanisms.

Tau phosphorylation and kinase activation in familial tauopathy linked to deln296 mutation.

PubMed

Ferrer, I; Pastor, P; Rey, M J; Muñoz, E; Puig, B; Pastor, E; Oliva, R; Tolosa, E

2003-02-01

Tau phosphorylation has been examined by immunohistochemistry in the brain of a patient affected with familial tauopathy with progressive supranuclear palsy-like phenotype linked to the delN296 mutation in the tau gene. Phospho-specific tau antibodies Thr181, Ser202, Ser214, Ser396 and Ser422, and antibodies to glycogen synthase kinase-3alpha/beta (GSK-3alpha/beta) and to phosphorylated (P) mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38 kinase (p38) and GSK-3betaSer9 have been used to gain understanding of the identification of phosphorylation sites, as well as of the specific kinases that regulate tau phosphorylation at those specific sites, in a familial tauopathy. The neuropathological examination disclosed atrophy of the right precentral gyrus and the brainstem. Neurone loss and gliosis were observed in the substantia nigra, several nuclei of the brainstem and diencephalon. Hyper-phosphorylated tau accumulated in neurones with neurofibrillary tangles and in neurones with pretangles in the substantia nigra, locus ceruleus, peri-aqueductal grey matter, reticular formation, motor nuclei of the brainstem, and thalamus, amygdala and hippocampus. tau-immunoreactive astrocytes and, particularly, oligodendrocytes with coiled bodies were widespread in the brainstem, diencephalons, cerebral white matter and cerebral cortex. Increased expression of MAPK/ERK-P, SAPK/JNK-P, p-38-P and GSK-3beta-P was observed in select subpopulations of neurones with neurofibrillary tangles and in neurones with pretangles. MAPK/ERK-P, SAPK/JNK-P, p38-P and GSK-3beta-P were also expressed in tau-containing astrocytes and in oligodendrocytes with coiled bodies. These findings show, for the first time, activation of precise kinases that regulate tau phosphorylation at specific sites in familial tauopathy.

Structural and mechanistic insights into Mps1 kinase activation.

PubMed

Wang, Wei; Yang, Yuting; Gao, Yuefeng; Xu, Quanbin; Wang, Feng; Zhu, Songcheng; Old, William; Resing, Katheryn; Ahn, Natalie; Lei, Ming; Liu, Xuedong

2009-08-01

Mps1 is one of the several essential kinases whose activation is required for robust mitotic spindle checkpoint signalling. The activity of Mps1 is tightly regulated and increases dramatically during mitosis or in response to spindle damage. To understand the molecular mechanism underlying Mps1 regulation, we determined the crystal structure of the kinase domain of Mps1. The 2.7-A-resolution crystal structure shows that the Mps1 kinase domain adopts a unique inactive conformation. Intramolecular interactions between the key Glu residue in the C helix of the N-terminal lobe and the backbone amides in the catalytic loop lock the kinase in the inactive conformation. Autophosphorylation appears to be a priming event for kinase activation. We identified Mps1 autophosphorylation sites in the activation and the P+1 loops. Whereas activation loop autophosphorylation enhances kinase activity, autophosphorylation at the P+1 loop (T686) is associated with the active kinase. Mutation of T686 autophosphorylation site impairs both autophosphorylation and transphosphorylation. Furthermore, we demonstrated that phosphorylation of T676 may be a priming event for phosphorylation at T686. Finally, we identified two critical lysine residues in the loop between helices EF and F that are essential for substrate recruitment and maintaining high levels of kinase activity. Our studies reveal critical biochemical mechanisms for Mps1 kinase regulation.

Structural and mechanistic insights into Mps1 kinase activation

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

Wang, Wei; Yang, Yuting; Gao, Yuefeng

2010-11-05

Mps1 is one of the several essential kinases whose activation is required for robust mitotic spindle checkpoint signalling. The activity of Mps1 is tightly regulated and increases dramatically during mitosis or in response to spindle damage. To understand the molecular mechanism underlying Mps1 regulation, we determined the crystal structure of the kinase domain of Mps1. The 2.7-{angstrom}-resolution crystal structure shows that the Mps1 kinase domain adopts a unique inactive conformation. Intramolecular interactions between the key Glu residue in the {alpha}C helix of the N-terminal lobe and the backbone amides in the catalytic loop lock the kinase in the inactive conformation.more » Autophosphorylation appears to be a priming event for kinase activation. We identified Mps1 autophosphorylation sites in the activation and the P+1 loops. Whereas activation loop autophosphorylation enhances kinase activity, autophosphorylation at the P+1 loop (T686) is associated with the active kinase. Mutation of T686 autophosphorylation site impairs both autophosphorylation and transphosphorylation. Furthermore, we demonstrated that phosphorylation of T676 may be a priming event for phosphorylation at T686. Finally, we identified two critical lysine residues in the loop between helices {alpha}EF and {alpha}F that are essential for substrate recruitment and maintaining high levels of kinase activity. Our studies reveal critical biochemical mechanisms for Mps1 kinase regulation.« less

Oncoprotein protein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning

1999-01-01

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

A Tumor Cell-Selective Inhibitor of Mitogen-Activated Protein Kinase Phosphatases Sensitizes Breast Cancer Cells to Lymphokine-Activated Killer Cell Activity

PubMed Central

Kaltenmeier, Christof T.; Vollmer, Laura L.; Vernetti, Lawrence A.; Caprio, Lindsay; Davis, Keanu; Korotchenko, Vasiliy N.; Day, Billy W.; Tsang, Michael; Hulkower, Keren I.; Lotze, Michael T.

2017-01-01

Dual specificity mitogen-activated protein kinase (MAPK) phosphatases [dual specificity phosphatase/MAP kinase phosphatase (DUSP-MKP)] have been hypothesized to maintain cancer cell survival by buffering excessive MAPK signaling caused by upstream activating oncogenic products. A large and diverse body of literature suggests that genetic depletion of DUSP-MKPs can reduce tumorigenicity, suggesting that hyperactivating MAPK signaling by DUSP-MKP inhibitors could be a novel strategy to selectively affect the transformed phenotype. Through in vivo structure-activity relationship studies in transgenic zebrafish we recently identified a hyperactivator of fibroblast growth factor signaling [(E)-2-benzylidene-5-bromo-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI-215)] that is devoid of developmental toxicity and restores defective MAPK activity caused by overexpression of DUSP1 and DUSP6 in mammalian cells. Here, we hypothesized that BCI-215 could selectively affect survival of transformed cells. In MDA-MB-231 human breast cancer cells, BCI-215 inhibited cell motility, caused apoptosis but not primary necrosis, and sensitized cells to lymphokine-activated killer cell activity. Mechanistically, BCI-215 induced rapid and sustained phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) in the absence of reactive oxygen species, and its toxicity was partially rescued by inhibition of p38 but not JNK or ERK. BCI-215 also hyperactivated MKK4/SEK1, suggesting activation of stress responses. Kinase phosphorylation profiling documented BCI-215 selectively activated MAPKs and their downstream substrates, but not receptor tyrosine kinases, SRC family kinases, AKT, mTOR, or DNA damage pathways. Our findings support the hypothesis that BCI-215 causes selective cancer cell cytotoxicity in part through non-redox-mediated activation of MAPK signaling, and the findings also identify an intersection with immune cell killing that is

A peptide fragment of ependymin neurotrophic factor uses protein kinase C and the mitogen-activated protein kinase pathway to activate c-Jun N-terminal kinase and a functional AP-1 containing c-Jun and c-Fos proteins in mouse NB2a cells.

PubMed

Adams, David S; Hasson, Brendan; Boyer-Boiteau, Anne; El-Khishin, Adam; Shashoua, Victor E

2003-05-01

Ependymin (EPN) is a goldfish brain neurotrophic factor previously shown to function in a variety of cellular events related to long-term memory formation and neuronal regeneration. CMX-8933, an 8-amino-acid synthetic peptide fragment of EPN, was designed for aiding an investigation of the biological properties of this glycoprotein. We reported from previous studies that treatment of mouse neuroblastoma (NB2a) cultures with CMX-8933 promotes activation of transcription factor AP-1, a characteristic previously associated with the following full-length neurotrophic factors: nerve growth factor, neurotropin-3, and brain-derived neurotrophic factor. The CMX-8933-activated AP-1 specifically bound an AP-1 consensus probe and appeared to contain c-Jun and c-Fos protein components in antibody supershift experiments. Because AP-1 influences a variety of positive and negative cellular processes, determined in part by its exact protein composition and mechanism of activation, we extended these initial AP-1 observations in the current study to confirm the identity of the CMX-8933-activated c-Jun and c-Fos components. CMX-8933 increases the enzymatic activity of c-Jun N-terminal kinase (JNK), increases the phosphorylation of JNK and c-Jun proteins, and increases the cellular titers of c-Jun and c-Fos mRNAs. Furthermore, the AP-1 activated by CMX-8933 is functional, insofar as it transactivates both synthetic and natural AP-1-dependent reporter plasmids. Inhibition studies indicate that activation of the 8933-induced AP-1 occurs via the mitogen-activated protein kinase pathway. These data are in agreement with the recently proposed model for the conversion of short- to long-term synaptic plasticity and memory, in which a JNK-activated transcription factor AP-1, containing c-Jun and c-Fos components, functions at the top of a hierarchy of transcription factors known to regulate long-term neural plasticity. Copyright 2003 Wiley-Liss, Inc.

Protection against β-amyloid neurotoxicity by a non-toxic endogenous N-terminal β-amyloid fragment and its active hexapeptide core sequence.

PubMed

Forest, Kelly H; Alfulaij, Naghum; Arora, Komal; Taketa, Ruth; Sherrin, Tessi; Todorovic, Cedomir; Lawrence, James L M; Yoshikawa, Gene T; Ng, Ho-Leung; Hruby, Victor J; Nichols, Robert A

2018-01-01

High levels (μM) of beta amyloid (Aβ) oligomers are known to trigger neurotoxic effects, leading to synaptic impairment, behavioral deficits, and apoptotic cell death. The hydrophobic C-terminal domain of Aβ, together with sequences critical for oligomer formation, is essential for this neurotoxicity. However, Aβ at low levels (pM-nM) has been shown to function as a positive neuromodulator and this activity resides in the hydrophilic N-terminal domain of Aβ. An N-terminal Aβ fragment (1-15/16), found in cerebrospinal fluid, was also shown to be a highly active neuromodulator and to reverse Aβ-induced impairments of long-term potentiation. Here, we show the impact of this N-terminal Aβ fragment and a shorter hexapeptide core sequence in the Aβ fragment (Aβcore: 10-15) to protect or reverse Aβ-induced neuronal toxicity, fear memory deficits and apoptotic death. The neuroprotective effects of the N-terminal Aβ fragment and Aβcore on Aβ-induced changes in mitochondrial function, oxidative stress, and apoptotic neuronal death were demonstrated via mitochondrial membrane potential, live reactive oxygen species, DNA fragmentation and cell survival assays using a model neuroblastoma cell line (differentiated NG108-15) and mouse hippocampal neuron cultures. The protective action of the N-terminal Aβ fragment and Aβcore against spatial memory processing deficits in amyloid precursor protein/PSEN1 (5XFAD) mice was demonstrated in contextual fear conditioning. Stabilized derivatives of the N-terminal Aβcore were also shown to be fully protective against Aβ-triggered oxidative stress. Together, these findings indicate an endogenous neuroprotective role for the N-terminal Aβ fragment, while active stabilized N-terminal Aβcore derivatives offer the potential for therapeutic application. © 2017 International Society for Neurochemistry.

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

PubMed Central

Andrusiak, Matthew G.; Jin, Yishi

2016-01-01

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

Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases

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

Marcotte, Douglas J.; Liu, Yu-Ting; Arduini, Robert M.

Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 {angstrom} resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 {angstrom} resolution. This data providesmore » information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.« less

Evidence for ACD5 ceramide kinase activity involvement in Arabidopsis response to cold stress.

PubMed

Dutilleul, Christelle; Chavarria, Heidy; Rézé, Nathalie; Sotta, Bruno; Baudouin, Emmanuel; Guillas, Isabelle

2015-12-01

Although sphingolipids emerged as important signals for plant response to low temperature, investigations have been limited so far to the function of long-chain base intermediates. The formation and function of ceramide phosphates (Cer-Ps) in chilled Arabidopsis were explored. Cer-Ps were analysed by thin layer chromatography (TLC) following in vivo metabolic radiolabelling. Ceramide kinase activity, gene expression and growth phenotype were determined in unstressed and cold-stressed wild type (WT) and Arabidopsis ceramide kinase mutant acd5. A rapid and transient formation of Cer-P occurs in cold-stressed WT Arabidopsis plantlets and cultured cells, which is strongly impaired in acd5 mutant. Although concomitant, Cer-P formation is independent of long-chain base phosphate (LCB-P) formation. No variation of ceramide kinase activity was measured in vitro in WT plantlets upon cold stress but the activity in acd5 mutant was further reduced by cold stress. At the seedling stage, acd5 response to cold was similar to that of WT. Nevertheless, acd5 seed germination was hypersensitive to cold and abscisic acid (ABA), and ABA-dependent gene expression was modified in acd5 seeds when germinated at low temperature. Our data involve for the first time Cer-P and ACD5 in low temperature response and further underline the complexity of sphingolipid signalling operating during cold stress. © 2015 John Wiley & Sons Ltd.

Receptor-mediated protein kinase activation and the mechanism of transmembrane signaling in bacterial chemotaxis.

PubMed Central

Liu, Y; Levit, M; Lurz, R; Surette, M G; Stock, J B

1997-01-01

Chemotaxis responses of Escherichia coli and Salmonella are mediated by type I membrane receptors with N-terminal extracytoplasmic sensing domains connected by transmembrane helices to C-terminal signaling domains in the cytoplasm. Receptor signaling involves regulation of an associated protein kinase, CheA. Here we show that kinase activation by a soluble signaling domain construct involves the formation of a large complex, with approximately 14 receptor signaling domains per CheA dimer. Electron microscopic examination of these active complexes indicates a well defined bundle composed of numerous receptor filaments. Our findings suggest a mechanism for transmembrane signaling whereby stimulus-induced changes in lateral packing interactions within an array of receptor-sensing domains at the cell surface perturb an equilibrium between active and inactive receptor-kinase complexes within the cytoplasm. PMID:9405352

Oncoprotein protein kinase

DOEpatents

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

1997-02-25

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

Differential regulation of CD44 expression by lipopolysaccharide (LPS) and TNF-alpha in human monocytic cells: distinct involvement of c-Jun N-terminal kinase in LPS-induced CD44 expression.

PubMed

Gee, Katrina; Lim, Wilfred; Ma, Wei; Nandan, Devki; Diaz-Mitoma, Francisco; Kozlowski, Maya; Kumar, Ashok

2002-11-15

Alterations in the regulation of CD44 expression play a critical role in modulating cell adhesion, migration, and inflammation. LPS, a bacterial cell wall component, regulates CD44 expression and may modulate CD44-mediated biological effects in monocytic cells during inflammation and immune responses. In this study, we show that in normal human monocytes, LPS and LPS-induced cytokines IL-10 and TNF-alpha enhance CD44 expression. To delineate the mechanism underlying LPS-induced CD44 expression, we investigated the role of the mitogen-activated protein kinases (MAPKs), p38, p42/44 extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) by using their specific inhibitors. We demonstrate the involvement, at least in part, of p38 MAPK in TNF-alpha-induced CD44 expression in both monocytes and promonocytic THP-1 cells. However, neither p38 nor p42/44 MAPKs were involved in IL-10-induced CD44 expression in monocytes. To further dissect the TNF-alpha and LPS-induced signaling pathways regulating CD44 expression independent of IL-10-mediated effects, we used IL-10 refractory THP-1 cells as a model system. Herein, we show that CD44 expression induced by the LPS-mediated pathway predominantly involved JNK activation. This conclusion was based on results derived by transfection of THP-1 cells with a dominant-negative mutant of stress-activated protein/extracellular signal-regulated kinase kinase 1, and by exposure of cells to JNK inhibitors dexamethasone and SP600125. All these treatments prevented CD44 induction in LPS-stimulated, but not in TNF-alpha-stimulated, THP-1 cells. Furthermore, we show that CD44 induction may involve JNK-dependent early growth response gene activation in LPS-stimulated monocytic cells. Taken together, these results suggest a predominant role of JNK in LPS-induced CD44 expression in monocytic cells.

Withaferin A activates stress signalling proteins in high risk acute lymphoblastic leukemia

PubMed Central

Shi, Li-Huan; Wu, Xi-Jun; Liu, Jun-Shan; Gao, Yin-Bo

2015-01-01

Withaferin A, the principal bio-active component isolated from the Withaniasomnifera, has shown promising anti-leukemic activity in addition to anti-invasive and anti-metastatic activity. The present study demonstrates the effect of withaferin A on the cell cycle status and the phosphorylation/activation of proteins involved in signal transduction in t(4;11) and non-t(4;11) acute lymphoblastic leukemia (ALL) cell lines after treatment with withaferin A. The cells after treatment with the vehicle or 25 μM withaferin A for 1, 2, 4 and 8 h were examined using flow cytometric analysis. The results revealed that withaferin A treatment induced cell growth arrest at the S to G2/M phase transition of the cell cycle. Withaferin A treatment also induced the phosphorylation of stress signalling proteins, including the p38 mitogen-activated protein kinase, the c-Jun N-terminal kinase, c-Jun, the heat shock protein 27 and protein kinase B within 0 to 16 h. These results were observed using multiplex technology and Western blotting analysis. Thus withaferin A induces stress response leading to cell death. Therefore, withaferin A can be a potent therapeutic agent for the treatment of high risk ALL with chromosomal translocation t(4;11). PMID:26884834

Withaferin A activates stress signalling proteins in high risk acute lymphoblastic leukemia.

PubMed

Shi, Li-Huan; Wu, Xi-Jun; Liu, Jun-Shan; Gao, Yin-Bo

2015-01-01

Withaferin A, the principal bio-active component isolated from the Withaniasomnifera, has shown promising anti-leukemic activity in addition to anti-invasive and anti-metastatic activity. The present study demonstrates the effect of withaferin A on the cell cycle status and the phosphorylation/activation of proteins involved in signal transduction in t(4;11) and non-t(4;11) acute lymphoblastic leukemia (ALL) cell lines after treatment with withaferin A. The cells after treatment with the vehicle or 25 μM withaferin A for 1, 2, 4 and 8 h were examined using flow cytometric analysis. The results revealed that withaferin A treatment induced cell growth arrest at the S to G2/M phase transition of the cell cycle. Withaferin A treatment also induced the phosphorylation of stress signalling proteins, including the p38 mitogen-activated protein kinase, the c-Jun N-terminal kinase, c-Jun, the heat shock protein 27 and protein kinase B within 0 to 16 h. These results were observed using multiplex technology and Western blotting analysis. Thus withaferin A induces stress response leading to cell death. Therefore, withaferin A can be a potent therapeutic agent for the treatment of high risk ALL with chromosomal translocation t(4;11).

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

PubMed

Andrusiak, Matthew G; Jin, Yishi

2016-04-08

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

DNA replication checkpoint signaling depends on a Rad53-Dbf4 N-terminal interaction in Saccharomyces cerevisiae.

PubMed

Chen, Ying-Chou; Kenworthy, Jessica; Gabrielse, Carrie; Hänni, Christine; Zegerman, Philip; Weinreich, Michael

2013-06-01

Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53-Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4-Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53-Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.

Opposite Roles of RNase and Kinase Activities of Inositol-Requiring Enzyme 1 (IRE1) on HSV-1 Replication

PubMed Central

Su, Airong; Wang, Huanru; Li, Yanlei; Wang, Xiaohui; Chen, Deyan; Wu, Zhiwei

2017-01-01

In response to the endoplasmic reticulum (ER) stress induced by herpes simplex virus type 1 (HSV-1) infection, host cells activate the unfolded protein response (UPR) to reduce the protein-folding burden in the ER. The regulation of UPR upon HSV-1 infection is complex, and the downstream effectors can be detrimental to viral replication. Therefore, HSV-1 copes with the UPR to create a beneficial environment for its replication. UPR has three branches, including protein kinase RNA (PKR)-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activated transcription factor 6 (ATF6). IRE1α is the most conserved branch of UPR which has both RNase and kinase activities. Previous studies have shown that IRE1α RNase activity was inactivated during HSV-1 infection. However, the effect of the two activities of IRE1α on HSV-1 replication remains unknown. Results in this study showed that IRE1α expression was up-regulated during HSV-1 infection. We found that in HEC-1-A cells, increasing RNase activity, or inhibiting kinase activity of IRE1α led to viral suppression, indicating that the kinase activity of IRE1α was beneficial, while the RNase activity was detrimental to viral replication. Further evidence showed that the kinase activity of IRE1α leads to the activation of the JNK (c-Jun N-terminal kinases) pathway, which enhances viral replication. Taken together, our evidence suggests that IRE1α is involved in HSV-1 replication, and its RNase and kinase activities play differential roles during viral infection. PMID:28832521

An X-ray structural study of pyruvate dehydrogenase kinase: A eukaryotic serine kinase with a prokaryotic histidine-kinase fold

NASA Astrophysics Data System (ADS)

Steussy, Calvin Nicklaus, Jr.

2001-07-01

Pyruvate Dehydrogenase Kinase is an enzyme that controls the flow of glucose through the eukaryotic cell and contributes to the pathology of diabetes mellitus. Early work on this kinase demonstrated that it has an amino acid sequence much like bacterial histidine kinases, but an activity similar to that of modern serine/threonine kinases. This project utilized the techniques of X-ray crystallography to determine molecular structure of pyruvate dehydrogenase kinase, isozyme 2. The structure was phased using selenium substituted for sulfur in methionine residues, and data at multiple wavelengths was collected at the National Synchrotron Light Source, Brookhaven National Laboratories. PDK 2 was found to fold into a two-domain monomer that forms a dimer through two beta sheets in the C-terminal domain. The N-terminal domain is an alpha-helical bundle while the C-terminal domain is an alpha/beta sandwich. The fold of the C-terminal domain is very similar to that of the prokaryotic histidine kinases, indicating that they share a common ancestor. The catalytic mechanism, however, has evolved to use general base catalysis to activate the serine substrate, rather than the direct nucleophilic attack by the imidazole sidechain used in the prokaryotic kinases. Thus, the structure of the protein echoes its prokaryotic ancestor, while the chemical mechanism has adapted to a serine substrate. The electrostatic surface of PDK2 leads to the suggestion that the lipoyl domain of the pyruvate dehydrogenase kinase, an important associated structure, may bind in the cleft formed between the N- and C-terminal domains. In addition, a network of hydrogen bonds directly connects the nucleotide binding pocket to the dimer interface, suggesting that there may be some interaction between dimer formation and ATP binding or ADP release.

Cannabidiol causes activated hepatic stellate cell death through a mechanism of endoplasmic reticulum stress-induced apoptosis

PubMed Central

Lim, M P; Devi, L A; Rozenfeld, R

2011-01-01

The major cellular event in the development and progression of liver fibrosis is the activation of hepatic stellate cells (HSCs). Activated HSCs proliferate and produce excess collagen, leading to accumulation of scar matrix and fibrotic liver. As such, the induction of activated HSC death has been proposed as a means to achieve resolution of liver fibrosis. Here we demonstrate that cannabidiol (CBD), a major non-psychoactive component of the plant Cannabis sativa, induces apoptosis in activated HSCs through a cannabinoid receptor-independent mechanism. CBD elicits an endoplasmic reticulum (ER) stress response, characterized by changes in ER morphology and the initiation of RNA-dependent protein kinase-like ER kinase-, activating transcription factor-6-, and inositol-requiring ER-to-nucleus signal kinase-1 (IRE1)-mediated signaling cascades. Furthermore, CBD induces downstream activation of the pro-apoptotic IRE1/ASK1/c-Jun N-terminal kinase pathway, leading to HSC death. Importantly, we show that this mechanism of CBD-induced ER stress-mediated apoptosis is specific to activated HSCs, as it occurs in activated human and rat HSC lines, and in primary in vivo-activated mouse HSCs, but not in quiescent HSCs or primary hepatocytes from rat. Finally, we provide evidence that the elevated basal level of ER stress in activated HSCs has a role in their susceptibility to the pro-apoptotic effect of CBD. We propose that CBD, by selectively inducing death of activated HSCs, represents a potential therapeutic agent for the treatment of liver fibrosis. PMID:21654828

Downregulation of Cellular c-Jun N-Terminal Protein Kinase and NF-κB Activation by Berberine May Result in Inhibition of Herpes Simplex Virus Replication

PubMed Central

Song, Siwei; Qiu, Min; Chu, Ying; Chen, Deyan; Wang, Xiaohui; Su, Airong

2014-01-01

Berberine is a quaternary ammonium salt from the protoberberine group of isoquinoline alkaloids. Some reports show that berberine exhibits anti-inflammatory, antitumor, and antiviral properties by modulating multiple cellular signaling pathways, including p53, nuclear factor κB (NF-κB), and mitogen-activated protein kinase. In the present study, we investigated the antiviral effect of berberine against herpes simplex virus (HSV) infection. Current antiherpes medicines such as acyclovir can lessen the recurring activation when used early at infection but are unable to prevent or cure infections where treatment has selected for resistant mutants. In searching for new antiviral agents against herpesvirus infection, we found that berberine reduced viral RNA transcription, protein synthesis, and virus titers in a dose-dependent manner. To elucidate the mechanism of its antiviral activity, the effect of berberine on the individual steps of viral replication cycle of HSV was investigated via time-of-drug addition assay. We found that berberine acted at the early stage of HSV replication cycle, between viral attachment/entry and genomic DNA replication, probably at the immediate-early gene expression stage. We further demonstrated that berberine significantly reduced HSV-induced NF-κB activation, as well as IκB-α degradation and p65 nuclear translocation. Moreover, we found that berberine also depressed HSV-induced c-Jun N-terminal kinase (JNK) phosphorylation but had little effect on p38 phosphorylation. Our results suggest that the berberine inhibition of HSV infection may be mediated through modulating cellular JNK and NF-κB pathways. PMID:24913175

A combination of increased Rho kinase activity and N-terminal pro-B-type natriuretic peptide predicts worse cardiovascular outcome in patients with acute coronary syndrome

PubMed Central

Dong, Ming; Liao, James K.; Yan, Bryan; Li, Ruijie; Zhang, Mang; Yu, Cheuk-Man

2013-01-01

Background Recent experimental evidence suggests that the Rho/Rho-kinase (ROCK) system may play an important role in the pathogenesis of acute coronary syndrome (ACS) but there are little clinical data. This study examined if ROCK activity is increased in patients with acute coronary syndrome and if ROCK activity predicts long-term cardiovascular event. Method Blood samples were collected from 188 patients within 12 h after admission for ACS (53% men; aged 70±13) and from 61 control subject. The main outcome measures were all cause mortality, readmission with ACS or congestive heart failure (CHF) from presentation within around 2 years (mean:14.4±7.2 months; range: 0.5 to 26 months). Results ROCK activity increased in ST elevation myocardial infarction (STEMI, n=90) (3.33±0.93), non-STEMI (NSTEMI, n=68) (3.37±1.04) and unstable angina (UA, n=30) (2.53±0.59) groups when compared with disease controls (n=31) (2.06±0.38, all p<0.001) and healthy controls (n=30) (1.54±0.43, all p<0.001). There were 24 deaths, 34 readmissions with ACS and 15 admissions with CHF within 2 years. Patients with a high N-terminal pro-B-type natriuretic peptide (NT-proBNP) and high ROCK activity on admission had a five-fold risk of a cardiovascular event (RR: 5.156; 95% CI: 2.180–12.191) when compared to those with low NT-proBNP and low ROCK activity. Conclusion ROCK activity was increased in patients with ACS, particularly in those with myocardial infarction. The combined usage of both ROCK activity and NT-proBNP might identify a subset of ACS patients at particularly high risk. PMID:22921817

Identification and Characterization of a Novel Class of c-Jun N-terminal Kinase Inhibitors

PubMed Central

Schepetkin, Igor A.; Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Hanks, Tracey S.; Kochetkova, Irina; Pascual, David W.; Jutila, Mark A.

2012-01-01

In efforts to identify novel small molecules with anti-inflammatory properties, we discovered a unique series of tetracyclic indenoquinoxaline derivatives that inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 activation. Compound IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) was found to be a potent, noncytotoxic inhibitor of pro-inflammatory cytokine [interleukin (IL)-1α, IL-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, interferon-γ, and granulocyte-macrophage colony-stimulating factor] and nitric oxide production by human and murine monocyte/macrophages. Three additional potent inhibitors of cytokine production were identified through further screening of IQ-1 analogs. The sodium salt of IQ-1 inhibited LPS-induced TNF-α and IL-6 production in MonoMac-6 cells with IC50 values of 0.25 and 0.61 μM, respectively. Screening of 131 protein kinases revealed that derivative IQ-3 [11H-indeno[1,2-b]quinoxalin-11-one-O-(2-furoyl)oxime]was a specific inhibitor of the c-Jun N-terminal kinase (JNK) family, with preference for JNK3. This compound, as well as IQ-1 and three additional oxime indenoquinoxalines, were found to be high-affinity JNK inhibitors with nanomolar binding affinity and ability to inhibit c-Jun phosphorylation. Furthermore, docking studies showed that hydrogen bonding interactions of the active indenoquinoxalines with Asn152, Gln155, and Met149 of JNK3 played an important role in enzyme binding activity. Finally, we showed that the sodium salt of IQ-1 had favorable pharmacokinetics and inhibited the ovalbumin-induced CD4+ T-cell immune response in a murine delayed-type hypersensitivity model in vivo. We conclude that compounds with an indenoquinoxaline nucleus can serve as specific small-molecule modulators for mechanistic studies of JNKs as well as a potential leads for the development of anti-inflammatory drugs. PMID:22434859

Increased sensitivity to apoptosis induced by methotrexate is mediated by Jun N-terminal kinase

PubMed Central

Spurlock, Charles F.; Aune, Zachary T.; Tossberg, John T.; Collins, Patrick L.; Aune, Jessica P.; Huston, Joseph W.; Crooke, Philip S.; Olsen, Nancy J.; Aune, Thomas M.

2011-01-01

Objective Low-dose methotrexate [MTX] is an effective therapy for rheumatoid arthritis yet its mechanism of action is incompletely understood. Here, we explored induction of apoptosis by MTX. Methods We employed flow cytometry to assess changes in levels of intracellular proteins, reactive oxygen species [ROS], and apoptosis.Quantitative polymerase chain reaction was usedtoassess changes in transcript levels of select target genes in response to MTX. Results MTX does not directly induce apoptosis but rather ‘primes’ cells for markedly increased sensitivity to apoptosis via either mitochondrial or death receptor pathways by a Jun N-terminal kinase [JNK]-dependent mechanism. Increased sensitivity to apoptosis is mediated, at least in part, by MTX-dependent production of reactive oxygen species, JNK activation and JNK-dependent induction of genes whose protein products promote apoptosis. Supplementation with tetrahydrobiopterin blocks these methotrexate-induced effects. Subjects with rheumatoid arthritis on low-dose MTX therapy express elevated levels of the JNK-target gene, JUN. Conclusions Our results support a model whereby methotrexate inhibits reduction of dihydrobiopterin to tetrahydrobiopterin resulting in increased production of ROS, increased JNK activity and increased sensitivity to apoptosis. The finding of increased JUN levels in subjects with RA taking low-dose MTX supports the notion that this pathway is activated by MTX, in vivo, and may contribute to efficacy of MTX in inflammatory disease. PMID:21618198

Tangeretin reduces ultraviolet B (UVB)-induced cyclooxygenase-2 expression in mouse epidermal cells by blocking mitogen-activated protein kinase (MAPK) activation and reactive oxygen species (ROS) generation.

PubMed

Yoon, Ji Hye; Lim, Tae-Gyu; Lee, Kyung Mi; Jeon, Ae Ji; Kim, Su Yeon; Lee, Ki Won

2011-01-12

The present study examined the effects of tangeretin, a polymethoxylated flavonone present in citrus fruits, on ultraviolet B (UVB)-induced cyclooxygenase-2 (COX-2) expression in JB6 P+ mouse skin epidermal cells. Tangeretin suppressed UVB-induced COX-2 expression and transactivation of nuclear factor-κB and activator protein-1 in JB6 P+ cells. Moreover, tangeretin blocked UVB-induced phosphorylation of Akt and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38, and attenuated the phosphorylation of MAPK kinases 1/2, 3/6, and 4. Tangeretin also limited the endogenous generation of reactive oxygen species (ROS), thereby protecting the cells against oxidative stress. However, tangeretin did not scavenge the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and influence the nicotinamide adenine dinucleotide phosphate oxidase activity. These results suggest that the anti-inflammatory effects of tangeretin stem from its modulation of cell signaling and suppression of intracellular ROS generation. Tangeretin may have a potent chemopreventive effect in skin cancer.

Oncoprotein protein kinase antibody kit

DOEpatents

Karin, Michael [San Diego, CA; Hibi, Masahiko [San Diego, CA; Lin, Anning [La Jolla, CA

2008-12-23

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

Puerarin reduces apoptosis in rat hippocampal neurons culturea in high glucose medium by modulating the p38 mitogen activated protein kinase and c-Jun N-terminal kinase signaling pathways.

PubMed

Xu, Xiaohan; Wang, Jingbo; Zhang, Hong; Tian, Guoqing; Liu, Yuqin

2016-02-01

To investigate the neuroprotective etfect of puerarin on rat hippocampal neurons cultured in high glucose medium, and to examine the role of the p38 mitogen activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) signaling pathways in this effect. Primary cultures of hippocampal neurons were prepared from newborn Sprague Dawley rats. Neuron-specific enolase immunocytochemistry was used to identify neurons. The neurons were cultured with normal medium (control group) or with high-glucose medium (high-glucose group), and puerarin (puerarin group), a p38 MAPK inhibitor (SB239063; p38 MAPK inhibitor group) or a JNK inhibitor (SP600125; JNK inhibitor group) were added. After 72 h of treatment, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was performed to detect apoptosis, and western blotting was used to assess protein levels of p-p38, p38, p-JNK and JNK. In the high-glucose group, the neuronal apoptosis rate and the p-p38/p38 and p-JNK/JNK ratios were higher than in the control group. The p38 MAPK and JNK inhibitors prevented this increase in the apoptosis rate. The apoptosis rates in the puerarin group, the p38 MAPK inhibitor group and the JNK inhibitor group were significantly decreased compared with the high-glucose group. Moreover, protein levels of p-p38 and p-JNK were significantly reduced, and the p-p38/p38 and p-JNK/JNK ratios were decreased in the puerarin group compared with the high-glucose group. In addition, compared with the high-glucose group, p-p38 levels and the p-p38/p38 ratio were reduced in the p38 MAPK inhibitor group, and p-JNK levels and the p-JNK/JNK ratio were decreased in the JNK inhibitor group. Puerarin attenuates neuronal apoptosis induced by high glucose by reducing the phosphorylation of p38 and JNK.

The drosophila T-box transcription factor midline functions within Insulin/Akt and c-Jun-N terminal kinase stress-reactive signaling pathways to regulate interommatial bristle formation and cell survival

PubMed Central

Chen, Q. Brent; Das, Sudeshna; Visic, Petra; Buford, Kendrick D.; Zong, Yan; Buti, Wisam; Odom, Kelly R.; Lee, Hannah; Leal, Sandra M.

2015-01-01

We recently reported that the T-box transcription factor midline (mid) functions within the Notch-Delta signaling pathway to specify sensory organ precursor (SOP) cell fates in early-staged pupal eye imaginal discs and to suppress apoptosis (Das et al.). From genetic and allelic modifier screens, we now report that mid interacts with genes downstream of the insulin receptor(InR)/Akt, c-Jun-N-terminal kinase (JNK) and Notch signaling pathways to regulate interommatidial bristle (IOB) formation and cell survival. One of the most significant mid-interacting genes identified from the modifier screen is dFOXO, a transcription factor exhibiting a nucleocytoplasmic subcellular distribution pattern. In common with dFOXO, we show that Mid exhibits a nucleocytoplasmic distribution pattern within WT third-instar larval (3°L) tissue homogenates. Because dFOXO is a stress-responsive factor, we assayed the effects of either oxidative or metabolic stress responses on modifying the mid mutant phenotype which is characterized by a 50% loss of IOBs within the adult compound eye. While metabolic starvation stress does not affect the mid mutant phenotype, either 1 mM paraquat or 20% coconut oil, oxidative stress inducers, partially suppresses the mid mutant phenotype resulting in a significant recovery of IOBs. Another significant mid-interacting gene we identified is groucho (gro). Mid and Gro are predicted to act as corepressors of the enhancer-of-split gene complex downstream of Notch. Immunolabeling WT and dFOXO null 3°L eye-antennal imaginal discs with anti-Mid and anti-Engrailed (En) antibodies indicate that dFOXO is required to activate Mid and En expression within photoreceptor neurons of the eye disc. Taken together, these studies show that Mid and dFOXO serve as critical effectors of cell fate specification and survival within integrated Notch, InR/dAkt, and JNK signaling pathways during 3°L and pupal eye imaginal disc development. PMID:25748605

AMP-activated protein kinase, stress responses and cardiovascular diseases

PubMed Central

WANG, Shaobin; SONG, Ping; ZOU, Ming-Hui

2012-01-01

AMPK (AMP-activated protein kinase) is one of the key players in maintaining intracellular homoeostasis. AMPK is well known as an energy sensor and can be activated by increased intracellular AMP levels. Generally, the activation of AMPK turns on catabolic pathways that generate ATP, while inhibiting cell proliferation and biosynthetic processes that consume ATP. In recent years, intensive investigations on the regulation and the function of AMPK indicates that AMPK not only functions as an intracellular energy sensor and regulator, but is also a general stress sensor that is important in maintaining intracellular homoeostasis during many kinds of stress challenges. In the present paper, we will review recent literature showing that AMPK functions far beyond its proposed energy sensor and regulator function. AMPK regulates ROS (reactive oxygen species)/redox balance, autophagy, cell proliferation, cell apoptosis, cellular polarity, mitochondrial function and genotoxic response, either directly or indirectly via numerous downstream pathways under physiological and pathological conditions. PMID:22390198

Evidence that N-acetylcysteine inhibits TNF-alpha-induced cerebrovascular endothelin-1 upregulation via inhibition of mitogen- and stress-activated protein kinase.

PubMed

Sury, Matthias D; Frese-Schaper, Manuela; Mühlemann, Miranda K; Schulthess, Fabienne T; Blasig, Ingolf E; Täuber, Martin G; Shaw, Sidney G; Christen, Stephan

2006-11-01

N-acetylcysteine (NAC) is neuroprotective in animal models of acute brain injury such as caused by bacterial meningitis. However, the mechanism(s) by which NAC exerts neuroprotection is unclear. Gene expression of endothelin-1 (ET-1), which contributes to cerebral blood flow decline in acute brain injury, is partially regulated by reactive oxygen species, and thus a potential target of NAC. We therefore examined the effect of NAC on tumor necrosis factor (TNF)-alpha-induced ET-1 production in cerebrovascular endothelial cells. NAC dose dependently inhibited TNF-alpha-induced preproET-1 mRNA upregulation and ET-1 protein secretion, while upregulation of inducible nitric oxide synthase (iNOS) was unaffected. Intriguingly, NAC had no effect on the initial activation (i.e., IkappaB degradation, nuclear p65 translocation, and Ser536 phosphorylation) of NF-kappaB by TNF-alpha. However, transient inhibition of NF-kappaB DNA binding suggested that NAC may inhibit ET-1 upregulation by inhibiting (a) parallel pathway(s) necessary for full transcriptional activation of NF-kappaB-mediated ET-1 gene expression. Similar to NAC, the MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and the protein kinase inhibitor H-89 selectively inhibited ET-1 upregulation without affecting nuclear p65 translocation, suggesting that NAC inhibits ET-1 upregulation via inhibition of mitogen- and stress-activated protein kinase (MSK). Supporting this notion, cotreatment with NAC inhibited the TNF-alpha-induced rise in MSK1 and MSK2 kinase activity, while siRNA knock-down experiments showed that MSK2 is the predominant isoform involved in TNF-alpha-induced ET-1 upregulation.

DNA Replication Checkpoint Signaling Depends on a Rad53–Dbf4 N-Terminal Interaction in Saccharomyces cerevisiae

PubMed Central

Chen, Ying-Chou; Kenworthy, Jessica; Gabrielse, Carrie; Hänni, Christine; Zegerman, Philip; Weinreich, Michael

2013-01-01

Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53–Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4–Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53–Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity. PMID:23564203

Oxidative Unfolding of the Rubredoxin Domain and the Natively Disordered N-terminal Region Regulate the Catalytic Activity of Mycobacterium tuberculosis Protein Kinase G*

PubMed Central

Wittwer, Matthias; Luo, Qi; Kaila, Ville R. I.

2016-01-01

Mycobacterium tuberculosis escapes killing in human macrophages by secreting protein kinase G (PknG). PknG intercepts host signaling to prevent fusion of the phagosome engulfing the mycobacteria with the lysosome and, thus, their degradation. The N-terminal NORS (no regulatory secondary structure) region of PknG (approximately residues 1–75) has been shown to play a role in PknG regulation by (auto)phosphorylation, whereas the following rubredoxin-like metal-binding motif (RD, residues ∼74–147) has been shown to interact tightly with the subsequent catalytic domain (approximately residues 148–420) to mediate its redox regulation. Deletions or mutations in NORS or the redox-sensitive RD significantly decrease PknG survival function. Based on combined NMR spectroscopy, in vitro kinase assay, and molecular dynamics simulation data, we provide novel insights into the regulatory roles of the N-terminal regions. The NORS region is indeed natively disordered and rather dynamic. Consistent with most earlier data, autophosphorylation occurs in our assays only when the NORS region is present and, thus, in the NORS region. Phosphorylation of it results only in local conformational changes and does not induce interactions with the subsequent RD. Although the reduced, metal-bound RD makes tight interactions with the following catalytic domain in the published crystal structures, it can also fold in its absence. Our data further suggest that oxidation-induced unfolding of the RD regulates substrate access to the catalytic domain and, thereby, PknG function under different redox conditions, e.g. when exposed to increased levels of reactive oxidative species in host macrophages. PMID:27810897

Oxidative Unfolding of the Rubredoxin Domain and the Natively Disordered N-terminal Region Regulate the Catalytic Activity of Mycobacterium tuberculosis Protein Kinase G.

PubMed

Wittwer, Matthias; Luo, Qi; Kaila, Ville R I; Dames, Sonja A

2016-12-30

Mycobacterium tuberculosis escapes killing in human macrophages by secreting protein kinase G (PknG). PknG intercepts host signaling to prevent fusion of the phagosome engulfing the mycobacteria with the lysosome and, thus, their degradation. The N-terminal NORS (no regulatory secondary structure) region of PknG (approximately residues 1-75) has been shown to play a role in PknG regulation by (auto)phosphorylation, whereas the following rubredoxin-like metal-binding motif (RD, residues ∼74-147) has been shown to interact tightly with the subsequent catalytic domain (approximately residues 148-420) to mediate its redox regulation. Deletions or mutations in NORS or the redox-sensitive RD significantly decrease PknG survival function. Based on combined NMR spectroscopy, in vitro kinase assay, and molecular dynamics simulation data, we provide novel insights into the regulatory roles of the N-terminal regions. The NORS region is indeed natively disordered and rather dynamic. Consistent with most earlier data, autophosphorylation occurs in our assays only when the NORS region is present and, thus, in the NORS region. Phosphorylation of it results only in local conformational changes and does not induce interactions with the subsequent RD. Although the reduced, metal-bound RD makes tight interactions with the following catalytic domain in the published crystal structures, it can also fold in its absence. Our data further suggest that oxidation-induced unfolding of the RD regulates substrate access to the catalytic domain and, thereby, PknG function under different redox conditions, e.g. when exposed to increased levels of reactive oxidative species in host macrophages. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Ampelopsin-induced reactive oxygen species enhance the apoptosis of colon cancer cells by activating endoplasmic reticulum stress-mediated AMPK/MAPK/XAF1 signaling

PubMed Central

Park, Ga Bin; Jeong, Jee-Yeong; Kim, Daejin

2017-01-01

Ampelopsin (Amp) is bioactive natural product and exerts anti-cancer effects against several cancer types. The present study investigated the anti-colon cancer activity of Amp and explored its mechanism of action. The treatment of colon cancer cells with Amp resulted in the dose- and time-dependent induction of apoptosis via the activation of endoplasmic reticulum (ER) stress, 5′ adenosine monophosphate-activated protein kinase (AMPK), and c-Jun N-terminal protein kinase (JNK)/p38 mitogen-activated protein kinases (MAPKs). Salubrinal, an ER stress inhibitor, prevented the upregulation of ER stress-associated proteins, including phosphorylated protein kinase RNA-like ER kinase, phosphorylated eukaryotic translation initiation factor 2α, glucose-regulated protein 78, and CCAAT/enhancer-binding protein homologous protein, as well as suppressing AMPK activation and the MAPK signaling pathway. Knockdown of AMPK by RNA interference failed to block ER stress. Additionally, SP600125 (a JNK inhibitor) and SB203580 (a p38-MAPK inhibitor) effectively inhibited apoptosis and attenuated the expression of X-linked IAP-associated factor 1 (XAF1) and apoptotic Bcl-2 family proteins (BCL2 antagonist/killer 1 and BCL2-associated X protein) in Amp-treated colon cancer cells. Furthermore, reactive oxygen species (ROS)-mediated ER stress/AMPK apoptotic signaling pathway in Amp-treated colon cancer cells were markedly inhibited by treatment with N-acetyl-L-cysteine, a ROS scavenger. These results demonstrate that treatment with Amp induces the apoptotic death of colon cancer cells through ER stress-initiated AMPK/MAPK/XAF1 signaling. These results also provide experimental information for developing Amp as therapeutic drug against colon cancer. PMID:29250183

Uncaria rhynchophylla and Rhynchophylline inhibit c-Jun N-terminal kinase phosphorylation and nuclear factor-kappaB activity in kainic acid-treated rats.

PubMed

Hsieh, Ching-Liang; Ho, Tin-Yun; Su, Shan-Yu; Lo, Wan-Yu; Liu, Chung-Hsiang; Tang, Nou-Ying

2009-01-01

Our previous studies have shown that Uncaria rhynchophylla (UR) can reduce epileptic seizures. We hypothesized that UR and its major component rhynchophylline (RH), reduce epileptic seizures in rats treated with kainic acid (KA) by inhibiting nuclear factor-kappaB (NF-kappaB) and activator-protein-1 (AP-1) activity, and by eliminating superoxide anions. Therefore, the level of superoxide anions and the DNA binding activities of NF-kappaB and AP-1 were measured. Sprague-Dawley (SD) rats were pre-treated with UR (1.0 g/kg, i.p.), RH (0.25 mg/kg, i.p.), or valproic acid (VA, 250 mg/kg, i.p.) for 3 days and then KA was administered intra-peritoneal (i.p.). The results indicated that UR, RH, and VA can reduce epileptic seizures and the level of superoxide anions in the blood. Furthermore, KA was demonstrated to induce the DNA binding activities of NF-kappaB and AP-1. However, these inductions were inhibited by pre-treatment with UR, RH, or VA for 3 days. Moreover, UR and RH were shown to be involved in the suppression of c-Jun N-terminal kinase (JNK) phosphorylation. This study suggested that UR and RH have antiepileptic effects in KA-induced seizures and are associated with the regulation of the innate immune system via a reduction in the level of superoxide anions, JNK phosphorylation, and NF-kappaB activation.

The N-terminal domain of Slack determines the formation and trafficking of Slick/Slack heteromeric sodium-activated potassium channels.

PubMed

Chen, Haijun; Kronengold, Jack; Yan, Yangyang; Gazula, Valeswara-Rao; Brown, Maile R; Ma, Liqun; Ferreira, Gonzalo; Yang, Youshan; Bhattacharjee, Arin; Sigworth, Fred J; Salkoff, Larry; Kaczmarek, Leonard K

2009-04-29

Potassium channels activated by intracellular Na(+) ions (K(Na)) play several distinct roles in regulating the firing patterns of neurons, and, at the single channel level, their properties are quite diverse. Two known genes, Slick and Slack, encode K(Na) channels. We have now found that Slick and Slack subunits coassemble to form heteromeric channels that differ from the homomers in their unitary conductance, kinetic behavior, subcellular localization, and response to activation of protein kinase C. Heteromer formation requires the N-terminal domain of Slack-B, one of the alternative splice variants of the Slack channel. This cytoplasmic N-terminal domain of Slack-B also facilitates the localization of heteromeric K(Na) channels to the plasma membrane. Immunocytochemical studies indicate that Slick and Slack-B subunits are coexpressed in many central neurons. Our findings provide a molecular explanation for some of the diversity in reported properties of neuronal K(Na) channels.

Primate Torpor: Regulation of Stress-activated Protein Kinases During Daily Torpor in the Gray Mouse Lemur, Microcebus murinus.

PubMed

Biggar, Kyle K; Wu, Cheng-Wei; Tessier, Shannon N; Zhang, Jing; Pifferi, Fabien; Perret, Martine; Storey, Kenneth B

2015-04-01

Very few selected species of primates are known to be capable of entering torpor. This exciting discovery means that the ability to enter a natural state of dormancy is an ancestral trait among primates and, in phylogenetic terms, is very close to the human lineage. To explore the regulatory mechanisms that underlie primate torpor, we analyzed signal transduction cascades to discover those involved in coordinating tissue responses during torpor. The responses of mitogen-activated protein kinase (MAPK) family members to primate torpor were compared in six organs of control (aroused) versus torpid gray mouse lemurs, Microcebus murinus. The proteins examined include extracellular signal-regulated kinases (ERKs), c-jun NH2-terminal kinases (JNKs), MAPK kinase (MEK), and p38, in addition to stress-related proteins p53 and heat shock protein 27 (HSP27). The activation of specific MAPK signal transduction pathways may provide a mechanism to regulate the expression of torpor-responsive genes or the regulation of selected downstream cellular processes. In response to torpor, each MAPK subfamily responded differently during torpor and each showed organ-specific patterns of response. For example, skeletal muscle displayed elevated relative phosphorylation of ERK1/2 during torpor. Interestingly, adipose tissues showed the highest degree of MAPK activation. Brown adipose tissue displayed an activation of ERK1/2 and p38, whereas white adipose tissue showed activation of ERK1/2, p38, MEK, and JNK during torpor. Importantly, both adipose tissues possess specialized functions that are critical for torpor, with brown adipose required for non-shivering thermogenesis and white adipose utilized as the primary source of lipid fuel for torpor. Overall, these data indicate crucial roles of MAPKs in the regulation of primate organs during torpor. Copyright © 2015. Production and hosting by Elsevier Ltd.

Serine Phosphorylation of the Insulin-like Growth Factor I (IGF-1) Receptor C-terminal Tail Restrains Kinase Activity and Cell Growth*

PubMed Central

Kelly, Geraldine M.; Buckley, Deirdre A.; Kiely, Patrick A.; Adams, David R.; O'Connor, Rosemary

2012-01-01

Insulin-like growth factor I receptor (IGF-1R) signaling is essential for cell, organ, and animal growth. The C-terminal tail of the IGF-1R exhibits regulatory function, but the mechanism is unknown. Here, we show that mutation of Ser-1248 (S1248A) enhances IGF-1R in vitro kinase activity, autophosphorylation, Akt/mammalian target of rapamycin activity, and cell growth. Ser-1248 phosphorylation is mediated by GSK-3β in a mechanism that involves a priming phosphorylation on Ser-1252. GSK-3β knock-out cells exhibit reduced IGF-1R cell surface expression, enhanced IGF-1R kinase activity, and signaling. Examination of crystallographic structures of the IGF-1R kinase domain revealed that the 1248SFYYS1252 motif adopts a conformation tightly packed against the kinase C-lobe when Ser-1248 is in the unphosphorylated state that favors kinase activity. S1248A mutation is predicted to lock the motif in this position. In contrast, phosphorylation of Ser-1248 will drive profound structural transition of the sequence, critically affecting connection of the C terminus as well as exposing potential protein docking sites. Decreased kinase activity of a phosphomimetic S1248E mutant and enhanced kinase activity in mutants of its predicted target residue Lys-1081 support this auto-inhibitory model. Thus, the SFYYS motif controls the organization of the IGF-1R C terminus relative to the kinase domain. Its phosphorylation by GSK-3β restrains kinase activity and regulates receptor trafficking and signaling. PMID:22685298

Fluid Shear Stress-Induced JNK Activity Leads to Actin Remodeling for Cell Alignment

PubMed Central

Mengistu, Meron; Brotzman, Hannah; Ghadiali, Samir; Lowe-Krentz, Linda

2012-01-01

Fluid shear stress (FSS) exerted on endothelial cell surfaces induces actin cytoskeleton remodeling through mechanotransduction. This study was designed to determine whether FSS activates Jun N-terminal kinase (JNK), to examine the spatial and temporal distribution of active JNK relative to the actin cytoskeleton in endothelial cells exposed to different FSS conditions, and to evaluate the effects of active JNK on actin realignment. Exposure to 15 and 20 dyn/cm2 FSS induced higher activity levels of JNK than the lower 2 and 4 dyn/cm2 flow conditions. At the higher FSS treatments, JNK activity increased with increasing exposure time, peaking 30 minutes after flow onset with an 8-fold activity increase compared to cells in static culture. FSS-induced phospho-JNK co-localized with actin filaments at cell peripheries, as well as with stress fibers. Pharmacologically blocking JNK activity altered FSS-induced actin structure and distribution as a response to FSS. Our results indicate that FSS-induced actin remodeling occurs in three phases, and that JNK plays a role in at least one, suggesting that this kinase activity is involved in mechanotransduction from the apical surface to the actin cytoskeleton in endothelial cells. PMID:20626006

Targeting the MEF2-Like Transcription Factor Smp1 by the Stress-Activated Hog1 Mitogen-Activated Protein Kinase

PubMed Central

Nadal, Eulàlia de; Casadomé, Laura; Posas, Francesc

2003-01-01

Exposure of Saccharomyces cerevisiae to increases in extracellular osmolarity activates the stress-activated Hog1 mitogen-activated protein kinase (MAPK), which is essential for cell survival upon osmotic stress. Yeast cells respond to osmotic stress by inducing the expression of a very large number of genes, and the Hog1 MAPK plays a critical role in gene transcription upon stress. To understand how Hog1 controls gene expression, we designed a genetic screen to isolate new transcription factors under the control of the MAPK and identified the MEF2-like transcription factor, Smp1, as a target for Hog1. Overexpression of SMP1 induced Hog1-dependent expression of osmoresponsive genes such as STL1, whereas smp1Δ cells were defective in their expression. Consistently, smp1Δ cells displayed reduced viability upon osmotic shock. In vivo coprecipitation and phosphorylation studies showed that Smp1 and Hog1 interact and that Smp1 is phosphorylated upon osmotic stress in a Hog1-dependent manner. Hog1 phosphorylated Smp1 in vitro at the C-terminal region. Phosphorylation of Smp1 by the MAPK is essential for its function, since a mutant allele unable to be phosphorylated by the MAPK displays impaired stress responses. Thus, our data indicate that Smp1 acts downstream of Hog1, controlling a subset of the responses induced by the MAPK. Moreover, Smp1 concentrates in the nucleus during the stationary phase, and the lack of SMP1 results in cells that lose viability in the stationary phase. Localization of Smp1 depends on HOG1, and consistently, hog1Δ cells also lose viability during this growth phase. These data suggest that Smp1 could be mediating a role for the Hog1 MAPK during the stationary phase. PMID:12482976

PAK2 is cleaved and activated during hyperosmotic shock-induced apoptosis via a caspase-dependent mechanism: evidence for the involvement of oxidative stress.

PubMed

Chan, W H; Yu, J S; Yang, S D

1999-03-01

Hyperosmotic shock elicits a stress response in mammalian cells and can lead to apoptotic cell death. In the present study, we report that hyperosmotic shock can induce activation of a 36 kDa kinase detected by an in-gel kinase assay in several cell types, including mouse Balb/c 3T3 fibroblasts, and human Hep 3B and A431 cells. This 36 kDa kinase can be recognized by an antibody against the C-terminal region of a family of p21Cdc42/Rac-activated kinases (PAKs) on immunoblot. Further studies with this antibody and a PAK2-specific antibody against the N-terminal region of PAK2 demonstrate that hyperosmotic shock can induce cleavage of PAK2 to generate a 36 kDa C-terminal catalytic fragment in cells. The cleavage and activation of PAK2 was found to be closely associated with both DNA fragmentation and activation of an ICE/CED-3 family cysteine protease termed caspase-3 in hyperosmotically shocked cells. Furthermore, pretreating the cells with two caspase inhibitors (Ac-DEVD-cho and Ac-YVAD-cmk) could inhibit both cleavage/activation of PAK2 and DNA fragmentation induced by hyperosmotic shock. Moreover, all these hyperosmotic shock-induced changes (i.e., activation of caspase-3, cleavage/activation of PAK2, and DNA fragmentation) in cells could be blocked by antioxidants such as ascorbic acid (vitamine C), alpha-tocopherol (vitamine E), dithiothreitol, beta-mercaptoethanol, and glutathione. Taken together, our results show that PAK2 is cleaved and activated via a caspase-dependent mechanism during hyperosmotic shock-induced apoptosis and suggest the involvement of antioxidant-preventable oxidative stress in inducing this process.

Phosphorylation of purified mitochondrial Voltage-Dependent Anion Channel by c-Jun N-terminal Kinase-3 modifies channel voltage-dependence.

PubMed

Gupta, Rajeev; Ghosh, Subhendu

2017-06-01

Voltage-Dependent Anion Channel (VDAC) phosphorylated by c-Jun N-terminal Kinase-3 (JNK3) was incorporated into the bilayer lipid membrane. Single-channel electrophysiological properties of the native and the phosphorylated VDAC were compared. The open probability versus voltage curve of the native VDAC displayed symmetry around the voltage axis, whereas that of the phosphorylated VDAC showed asymmetry. This result indicates that phosphorylation by JNK3 modifies voltage-dependence of VDAC.

The mitochondria of stallion spermatozoa are more sensitive than the plasmalemma to osmotic-induced stress: role of c-Jun N-terminal kinase (JNK) pathway.

PubMed

García, Beatriz Macías; Moran, Alvaro Miró; Fernández, Lauro González; Ferrusola, Cristina Ortega; Rodriguez, Antolin Morillo; Bolaños, Juan Maria Gallardo; da Silva, Carolina Maria Balao; Martínez, Heriberto Rodríguez; Tapia, Jose A; Peña, Fernando J

2012-01-01

Cryopreservation introduces extreme temperature and osmolality changes that impart lethal and sublethal effects on spermatozoa. Additionally, there is evidence that the osmotic stress induced by cryopreservation causes oxidative stress to spermatozoa. The main sources of reactive oxygen species in mammalian sperm are the mitochondria. In view of this, the aim of our study was to test whether or not osmotic stress was able to induce mitochondrial damage and to explore the osmotic tolerance of the mitochondria of stallion spermatozoa. Ejaculates from 7 stallions were subjected to osmolalities ranging from 75 to 1500 mOsm/kg, and the effect on sperm membrane integrity and mitochondrial membrane potential was studied. Additionally, the effects of changes in osmolality from hyposmotic to isosmotic and from hyperosmotic to isosmotic solutions were studied (osmotic excursions). The cellular volume of stallion spermatozoa under isosmotic conditions was 20.4 ± 0.33 μm(3). When exposed to low osmolality, the stallion spermatozoa behaved like a linear osmometer, whereas exposure to high osmolalities up to 900 mOsm/kg resulted in decreased sperm volume. Although sperm membranes were relatively resistant to changes in osmolality, mitochondrial membrane potential decreased when osmolalities were low or very high (10.7 ± 1.74 and 16.5 ± 1.70 at 75 and 150 mOsm/kg, respectively, and 13.1 ± 1.83 at 1500 mOsm/kg), whereas in isosmolar controls the percentage of stallion sperm mitochondria with a high membrane potential was 41.1 ± 1.69 (P < .01). Osmotic excursions induced greater damage than exposure of spermatozoa to a given nonphysiologic osmolality, and again the mitochondria were more prone to damage induced by osmotic excursions than was the sperm plasma membrane. In search of intracellular components that could mediate these changes, we have detected for the first time the c-Jun N-terminal kinase 1/2 in stallion spermatozoa, which are apparently involved in the

Acetylation within the N- and C-Terminal Domains of Src Regulates Distinct Roles of STAT3-Mediated Tumorigenesis.

PubMed

Huang, Chao; Zhang, Zhe; Chen, Lihan; Lee, Hank W; Ayrapetov, Marina K; Zhao, Ting C; Hao, Yimei; Gao, Jinsong; Yang, Chunzhang; Mehta, Gautam U; Zhuang, Zhengping; Zhang, Xiaoren; Hu, Guohong; Chin, Y Eugene

2018-06-01

Posttranslational modifications of mammalian c-Src N-terminal and C-terminal domains regulate distinct functions. Myristoylation of G 2 controls its cell membrane association and phosphorylation of Y419/Y527 controls its activation or inactivation, respectively. We provide evidence that Src-cell membrane association-dissociation and catalytic activation-inactivation are both regulated by acetylation. In EGF-treated cells, CREB binding protein (CBP) acetylates an N-terminal lysine cluster (K5, K7, and K9) of c-Src to promote dissociation from the cell membrane. CBP also acetylates the C-terminal K401, K423, and K427 of c-Src to activate intrinsic kinase activity for STAT3 recruitment and activation. N-terminal domain phosphorylation (Y14, Y45, and Y68) of STAT3 by c-Src activates transcriptionally active dimers of STAT3. Moreover, acetyl-Src translocates into nuclei, where it forms the Src-STAT3 enhanceosome for gene regulation and cancer cell proliferation. Thus, c-Src acetylation in the N-terminal and C-terminal domains play distinct roles in Src activity and regulation. Significance: CBP-mediated acetylation of lysine clusters in both the N-terminal and C-terminal regions of c-Src provides additional levels of control over STAT3 transcriptional activity. Cancer Res; 78(11); 2825-38. ©2018 AACR . ©2018 American Association for Cancer Research.

Inhibition of c-Jun N-terminal kinase activation reverses Alzheimer disease phenotypes in APPswe/PS1dE9 mice.

PubMed

Zhou, Qiong; Wang, Man; Du, Ying; Zhang, Wei; Bai, Miao; Zhang, Zhuo; Li, Zhuyi; Miao, Jianting

2015-04-01

Growing evidence indicates that the activation of c-Jun N-terminal kinase (JNK) is implicated in the multiple major pathological features of Alzheimer disease (AD). However, whether specific inhibition of JNK activation could prevent disease progression in adult transgenic AD models at moderate stage remains unknown. Here we first investigated the potential disease-modifying therapeutic effect of systemic administration of SP600125, a small-molecule JNK-specific inhibitor, in middle-aged APPswe/PS1dE9 mice. Using behavioral, histological, and biochemical methods, outcomes of SP600125 treatment on neuropathology and cognitive deficits were studied in APPswe/PS1dE9 mice. Compared with vehicle-treated APPswe/PS1dE9 mice, chronic treatment of SP600125 for 12 weeks potently inhibited JNK activation, which resulted in a marked improvement of behavioral measures of cognitive deficits and a dramatic reduction in amyloid plaque burden, β-amyloid production, tau hyperphosphorylation, inflammatory responses, and synaptic loss in these transgenic animals. In particular, we found that SP600125 treatment strongly promoted nonamyloidogenic amyloid precursor protein (APP) processing and inhibited amyloidogenic APP processing via regulating APP-cleavage secretase expression (ie, ADAM10, BACE1, and PS1) in APPswe/PS1dE9 mice. Our findings demonstrate that chronic SP600125 treatment is powerfully effective in slowing down disease progression by markedly reducing multiple pathological features and ameliorating cognitive deficits associated with AD. This study highlights the concept that active JNK actually contributes to the development of the disease, and provides critical preclinical evidence that specific inhibition of JNK activation by SP600125 treatment may be a novel promising disease-modifying therapeutic strategy for the treatment of AD. © 2015 American Neurological Association.

Intramolecular activation of a Ca(2+)-dependent protein kinase is disrupted by insertions in the tether that connects the calmodulin-like domain to the kinase

NASA Technical Reports Server (NTRS)

Vitart, V.; Christodoulou, J.; Huang, J. F.; Chazin, W. J.; Harper, J. F.; Evans, M. L. (Principal Investigator)

2000-01-01

Ca(2+)-dependent protein kinases (CDPK) have a calmodulin-like domain (CaM-LD) tethered to the C-terminal end of the kinase. Activation is proposed to involve intramolecular binding of the CaM-LD to a junction sequence that connects the CaM-LD to the kinase domain. Consistent with this model, a truncated CDPK (DeltaNC) in which the CaM-LD has been deleted can be activated in a bimolecular interaction with an isolated CaM-LD or calmodulin, similar to the activation of a calmodulin-dependent protein kinase (CaMK) by calmodulin. Here we provide genetic evidence that this bimolecular activation requires a nine-residue binding segment from F436 to I444 (numbers correspond to CPK-1 accession number L14771). Two mutations at either end of this core segment (F436/A and VI444/AA) severely disrupted bimolecular activation, whereas flanking mutations had only minor effects. Intramolecular activation of a full-length kinase was also disrupted by a VI444/AA mutation, but surprisingly not by a F436/A mutation (at the N-terminal end of the binding site). Interestingly, intramolecular but not bimolecular activation was disrupted by insertion mutations placed immediately downstream of I444. To show that mutant enzymes were not misfolded, latent kinase activity was stimulated through binding of an antijunction antibody. Results here support a model of intramolecular activation in which the tether (A445 to G455) that connects the CaM-LD to the kinase provides an important structural constraint and is not just a simple flexible connection.

Cloning and characterization of microbial activated Aedes aegypti MEK4 (AaMEK4): influences of noncatalytic domains on enzymatic activity.

PubMed

Wu, R C-C; Cho, W-L

2014-10-01

Protein kinases are known to be involved in a number of signal transduction cascades. Both the stress-activated Jun N-terminal kinase (JNK) and mitogen-activated protein kinase (MAPK) p38 pathways have been shown to correlate with the insect immune response to microbial infection. MAP kinase kinase 4 (MEK4) is an upstream kinase of JNK and p38 kinase. The cDNA of AaMEK4 was cloned and characterized. AaMEK4 was activated by microbial lysates of Gram-positive, Gram-negative bacteria and yeast. The conserved lysine (K112 ) and the putative phosphorylation sites (S238 and T242 ) were shown to be important for kinase activity by site-directed mutagenesis. A common MAPK docking site (MAPK_dsA) was found and in addition, a new nearby docking site, MAPK_dsB, was identified in the N-terminal noncatalytic domain of AaMEK4. MAPK_dsB was shown to be a unique element in the MEK4 family. In this study, both MAPK_dsA and _dsB were demonstrated to be important to AaMEK4 enzymatic activity for the downstream protein kinase, Aap38. © 2014 The Royal Entomological Society.

Solution structure and backbone dynamics of the N-terminal region of the calcium regulatory domain from soybean calcium-dependent protein kinase alpha.

PubMed

Weljie, Aalim M; Gagné, Stéphane M; Vogel, Hans J

2004-12-07

Ca(2+)-dependent protein kinases (CDPKs) are vital Ca(2+)-signaling proteins in plants and protists which have both a kinase domain and a self-contained calcium regulatory calmodulin-like domain (CLD). Despite being very similar to CaM (>40% identity) and sharing the same fold, recent biochemical and structural evidence suggests that the behavior of CLD is distinct from its namesake, calmodulin. In this study, NMR spectroscopy is employed to examine the structure and backbone dynamics of a 168 amino acid Ca(2+)-saturated construct of the CLD (NtH-CLD) in which almost the entire C-terminal domain is exchange broadened and not visible in the NMR spectra. Structural characterization of the N-terminal domain indicates that the first Ca(2+)-binding loop is significantly more open than in a recently reported structure of the CLD complexed with a putative intramolecular binding region (JD) in the CDPK. Backbone dynamics suggest that parts of the third helix exhibit unusually high mobility, and significant exchange, consistent with previous findings that this helix interacts with the C-terminal domain. Dynamics data also show that the "tether" region, consisting of the first 11 amino acids of CLD, is highly mobile and these residues exhibit distinctive beta-type secondary structure, which may help to position the JD and CLD. Finally, the unusual global dynamic behavior of the protein is rationalized on the basis of possible interdomain rearrangements and the highly variable environments of the C- and N-terminal domains.

Momordica charantia polysaccharides could protect against cerebral ischemia/reperfusion injury through inhibiting oxidative stress mediated c-Jun N-terminal kinase 3 signaling pathway.

PubMed

Gong, Juanjuan; Sun, Fumou; Li, Yihang; Zhou, Xiaoling; Duan, Zhenzhen; Duan, Fugang; Zhao, Lei; Chen, Hansen; Qi, Suhua; Shen, Jiangang

2015-04-01

Momordica charantia (MC) is a medicinal plant for stroke treatment in Traditional Chinese Medicine, but its active compounds and molecular targets are unknown yet. M. charantia polysaccharide (MCP) is one of the important bioactive components in MC. In the present study, we tested the hypothesis that MCP has neuroprotective effects against cerebral ischemia/reperfusion injury through scavenging superoxide (O2(-)), nitric oxide (NO) and peroxynitrite (ONOO(-)) and inhibiting c-Jun N-terminal protein kinase (JNK3) signaling cascades. We conducted experiments with in vivo global and focal cerebral ischemia/reperfusion rat models and in vitro oxygen glucose deprivation (OGD) neural cells. The effects of MCP on apoptotic cell death and infarction volume, the bioactivities of scavenging O2(-), NO and ONOO(-), inhibiting lipid peroxidation and modulating JNK3 signaling pathway were investigated. Major results are summarized as below: (1) MCP dose-dependently attenuated apoptotic cell death in neural cells under OGD condition in vitro and reduced infarction volume in ischemic brains in vivo; (2) MCP had directing scavenging effects on NO, O2(-) and ONOO(-) and inhibited lipid peroxidation; (3) MCP inhibited the activations of JNK3/c-Jun/Fas-L and JNK3/cytochrome C/caspases-3 signaling cascades in ischemic brains in vivo. Taken together, we conclude that MCP could be a promising neuroprotective ingredient of M. charantia and its mechanisms could be at least in part attributed to its antioxidant activities and inhibiting JNK3 signaling cascades during cerebral ischemia/reperfusion injury. Copyright © 2014 Elsevier Ltd. All rights reserved.

A novel disulfide bond in the SH2 Domain of the C-terminal Src kinase controls catalytic activity.

PubMed

Mills, Jamie E; Whitford, Paul C; Shaffer, Jennifer; Onuchic, Jose N; Adams, Joseph A; Jennings, Patricia A

2007-02-02

The SH2 domain of the C-terminal Src kinase [Csk] contains a unique disulfide bond that is not present in other known SH2 domains. To investigate whether this unusual disulfide bond serves a novel function, the effects of disulfide bond formation on catalytic activity of the full-length protein and on the structure of the SH2 domain were investigated. The kinase activity of full-length Csk decreases by an order of magnitude upon formation of the disulfide bond in the distal SH2 domain. NMR spectra of the fully oxidized and fully reduced SH2 domains exhibit similar chemical shift patterns and are indicative of similar, well-defined tertiary structures. The solvent-accessible disulfide bond in the isolated SH2 domain is highly stable and far from the small lobe of the kinase domain. However, reduction of this bond results in chemical shift changes of resonances that map to a cluster of residues that extend from the disulfide bond across the molecule to a surface that is in direct contact with the small lobe of the kinase domain in the intact molecule. Normal mode analyses and molecular dynamics calculations suggest that disulfide bond formation has large effects on residues within the kinase domain, most notably within the active-site cleft. Overall, the data indicate that reversible cross-linking of two cysteine residues in the SH2 domain greatly impacts catalytic function and interdomain communication in Csk.

The c-Jun N-terminal kinase pathway is critical for cell transformation by the latent membrane protein 1 of Epstein-Barr virus

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

Kutz, Helmut; Reisbach, Gilbert; Schultheiss, Ute

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) transforms cells activating signal transduction pathways such as NF-{kappa}B, PI3-kinase, or c-Jun N-terminal kinase (JNK). Here, we investigated the functional role of the LMP1-induced JNK pathway in cell transformation. Expression of a novel dominant-negative JNK1 allele caused a block of proliferation in LMP1-transformed Rat1 fibroblasts. The JNK-specific inhibitor SP600125 reproduced this effect in Rat1-LMP1 cells and efficiently interfered with proliferation of EBV-transformed lymphoblastoid cells (LCLs). Inhibition of the LMP1-induced JNK pathway in LCLs caused the downregulation of c-Jun and Cdc2, the essential G2/M cell cycle kinase, which was accompanied bymore » a cell cycle arrest of LCLs at G2/M phase transition. Moreover, SP600125 retarded tumor growth of LCLs in a xenograft model in SCID mice. Our data support a critical role of the LMP1-induced JNK pathway for proliferation of LMP1-transformed cells and characterize JNK as a potential target for intervention against EBV-induced malignancies.« less

Hsp90 N- and C-terminal double inhibition synergistically suppresses Bcr-Abl-positive human leukemia cells

PubMed Central

Chen, Xianling; Chen, Xiaole; Li, Ding; Fan, Yingjuan; Xu, Jianhua; Chen, Yuanzhong; Wu, Lixian

2017-01-01

Heat shock protein 90 (Hsp90) contains amino (N)–terminal domain, carboxyl(C)-terminal domain, and middle domains, which activate Hsp90 chaperone function cooperatively in tumor cells. One terminal occupancy might influence another terminal binding with inhibitor. The Bcr-Abl kinase is one of the Hsp90 clients implicated in the pathogenesis of chronic myeloid leukemia (CML). Present studies demonstrate that double inhibition of the N- and C-terminal termini can disrupt Hsp90 chaperone function synergistically, but not antagonistically, in Bcr-Abl-positive human leukemia cells. Furthermore, both the N-terminal inhibitor 17-AAG and the C-terminal inhibitor cisplatin (CP) have the capacity to suppress progenitor cells; however, only CP is able to inhibit leukemia stem cells (LSCs) significantly, which implies that the combinational treatment is able to suppress human leukemia in different mature states. PMID:28036294

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

PubMed

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.

Inhibition of the Jun N-Terminal Protein Kinase Pathway by SHIP-1, a Lipid Phosphatase That Interacts with the Adaptor Molecule Dok-3

PubMed Central

Robson, Jeffrey D.; Davidson, Dominique; Veillette, André

2004-01-01

Dok-3 is a Dok-related adaptor expressed in B cells and macrophages. Previously, we reported that Dok-3 is an inhibitor of B-cell activation in A20 B cells and that it associates with SHIP-1, a 5′ inositol-specific lipid phosphatase, as well as Csk, a negative regulator of Src kinases. Here, we demonstrate that Dok-3 suppresses B-cell activation by way of its interaction with SHIP-1, rather than Csk. Our biochemical analyses showed that the Dok-3-SHIP-1 complex acts by selectively inhibiting the B-cell receptor (BCR)-evoked activation of the Jun N-terminal protein kinase (JNK) cascade without affecting overall protein tyrosine phosphorylation or activation of previously described SHIP-1 targets like Btk and Akt/PKB. Studies of B cells derived from SHIP-1-deficient mice showed that BCR-triggered activation of JNK is enhanced in the absence of SHIP-1, implying that the Dok-3-SHIP-1 complex (or a related mechanism) is a physiological negative regulator of the JNK cascade in normal B cells. Together, these data elucidate the mechanism by which Dok-3 inhibits B-cell activation. Furthermore, they provide evidence that SHIP-1 can be a negative regulator of JNK signaling in B cells. PMID:14993273

Comparative Molecular Dynamics Simulations of Mitogen-Activated Protein Kinase-Activated Protein Kinase 5

PubMed Central

Lindin, Inger; Wuxiuer, Yimingjiang; Ravna, Aina Westrheim; Moens, Ugo; Sylte, Ingebrigt

2014-01-01

The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD) simulations of: (1) MK5 alone; (2) MK5 in complex with an inhibitor; and (3) MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS) calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding. PMID:24651460

Oncoprotein protein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Linn, Anning

1996-01-01

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

The Stress-Responsive dgk Gene from Streptococcus mutans Encodes a Putative Undecaprenol Kinase Activity

PubMed Central

Lis, Maciej; Kuramitsu, Howard K.

2003-01-01

We analyzed a previously constructed stress-sensitive Streptococcus mutans mutant Tn-1 strain resulting from disruption by transposon Tn916 of a gene encoding a protein exhibiting amino acid sequence similarity to the Escherichia coli diacylglycerol kinase. It was confirmed that the mutation led to significantly reduced lipid kinase activity, while expression of the intact gene on a plasmid restored both kinase activity and the wild-type phenotype. Further analysis revealed that the product of the dgk gene in S. mutans predominantly recognizes a lipid substrate other than diacylglycerol, most likely undecaprenol, as demonstrated by its efficient phosphorylation and the resistance of the product of the reaction to saponification. The physiological role of the product of the dgk gene as a putative undecaprenol kinase was further supported by a significantly higher sensitivity of the mutant to bacitracin compared with that of the parental strain. PMID:12654811

Comparative study of the efficacy of pulsed electromagnetic field and low level laser therapy on mitogen-activated protein kinases.

PubMed

El-Makakey, Ayman M; El-Sharaby, Radwa M; Hassan, Mohammed H; Balbaa, Alaa

2017-03-01

Mitogen-Activated Protein Kinases (MAPKs) consist of three major signaling members: extracellular signal-regulated kinase (ERK), p38 and C-JUN N-terminal kinase (JNK). We investigated physiological effects of Pulsed Electromagnetic Field Therapy (PEMFT) and Low Level Laser Therapy (LLLT) on human body, adopting the expression level of mitogen-activated protein kinases as an indicator via assessment of the activation levels of three major families of MAPKS, ERK, p38 and JNK in the peripheral lymphocytes of patients before and after the therapies. Assessment for the expression levels of MAPKs families' were done, in the peripheral lymphocytes of patients recently have appendectomy, using flow cytometric analysis of multiple signaling pathways, pre and post LLLT and PEMFT application (twice daily for 6 successive days) on the appendectomy wound. There were non-significant differences in the expression levels of MAPKs families' pre- therapies application. But there were significant increase in the ERK expression levels post application of LLLT compared to its pre application (p<0.01). Also, there was significant increase in the ERK, p38 and C-Jun N terminal expression level values post application of PEMFT compared to its pre application expression levels (p<0.01 for each). The present study demonstrates that PEMFT has a powerful healing effect more than LLLT as it increase the activation of ERK, P38 and C-Jun-N Terminal while LLLT only increase the activation of ERK. LLLT has more potent pain decreasing effect than PEMFT as it does not activate P38 pathway like PEMFT.

Cloning and characterization of full-length mouse thymidine kinase 2: the N-terminal sequence directs import of the precursor protein into mitochondria.

PubMed Central

Wang, L; Eriksson, S

2000-01-01

The subcellular localization of mitochondrial thymidine kinase (TK2) has been questioned, since no mitochondrial targeting sequences have been found in cloned human TK2 cDNAs. Here we report the cloning of mouse TK2 cDNA from a mouse full-length enriched cDNA library. The mouse TK2 cDNA codes for a protein of 270 amino acids, with a 40-amino-acid presumed N-terminal mitochondrial targeting signal. In vitro translation and translocation experiments with purified rat mitochondria confirmed that the N-terminal sequence directed import of the precursor TK2 into the mitochondrial matrix. A single 2.4 kb mRNA transcript was detected in most tissues examined, except in liver, where an additional shorter (1.0 kb) transcript was also observed. There was no correlation between the tissue distribution of TK2 activity and the expression of TK2 mRNA. Full-length mouse TK2 protein and two N-terminally truncated forms, one of which corresponds to the mitochondrial form of TK2 and a shorter form corresponding to the previously characterized recombinant human TK2, were expressed in Escherichia coli and affinity purified. All three forms of TK2 phosphorylated thymidine, deoxycytidine and 2'-deoxyuridine, but with different kinetic efficiencies. A number of cytostatic pyrimidine nucleoside analogues were also tested and shown to be good substrates for the various forms of TK2. The active form of full-length mouse TK2 was a dimer, as judged by Superdex 200 chromatography. These results enhance our understanding of the structure and function of TK2, and may help to explain the mitochondrial disorder, mitochondrial neurogastrointestinal encephalomyopathy. PMID:11023833

ERK and p38 MAPK-Activated Protein Kinases: a Family of Protein Kinases with Diverse Biological Functions

PubMed Central

Roux, Philippe P.; Blenis, John

2004-01-01

Conserved signaling pathways that activate the mitogen-activated protein kinases (MAPKs) are involved in relaying extracellular stimulations to intracellular responses. The MAPKs coordinately regulate cell proliferation, differentiation, motility, and survival, which are functions also known to be mediated by members of a growing family of MAPK-activated protein kinases (MKs; formerly known as MAPKAP kinases). The MKs are related serine/threonine kinases that respond to mitogenic and stress stimuli through proline-directed phosphorylation and activation of the kinase domain by extracellular signal-regulated kinases 1 and 2 and p38 MAPKs. There are currently 11 vertebrate MKs in five subfamilies based on primary sequence homology: the ribosomal S6 kinases, the mitogen- and stress-activated kinases, the MAPK-interacting kinases, MAPK-activated protein kinases 2 and 3, and MK5. In the last 5 years, several MK substrates have been identified, which has helped tremendously to identify the biological role of the members of this family. Together with data from the study of MK-knockout mice, the identities of the MK substrates indicate that they play important roles in diverse biological processes, including mRNA translation, cell proliferation and survival, and the nuclear genomic response to mitogens and cellular stresses. In this article, we review the existing data on the MKs and discuss their physiological functions based on recent discoveries. PMID:15187187

Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

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

Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty

2010-09-21

Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium bindingmore » triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.« less

Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS

PubMed Central

Jun, Jesse E.; Yang, Ming; Chen, Hang; Chakraborty, Arup K.

2013-01-01

Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation. PMID:23589333

Specific inhibition of c-Jun N-terminal kinase delays preterm labour and reduces mortality

PubMed Central

Pirianov, Grisha; MacIntyre, David A; Lee, Yun; Waddington, Simon N; Terzidou, Vasso; Mehmet, Huseyin; Bennett, Phillip R

2015-01-01

Preterm labour (PTL) is commonly associated with infection and/or inflammation. Lipopolysaccharide (LPS) from different bacteria can be used to independently or mutually activate Jun N-terminal kinase (JNK)/AP1- or NF-κB-driven inflammatory pathways that lead to PTL. Previous studies using Salmonella abortus LPS, which activates both JNK/AP-1 and NF-κB, showed that selective inhibition of NF-κB delays labour and improves pup outcome. Where labour is induced using Escherichia coli LPS (O111), which upregulates JNK/AP-1 but not NF-κB, inhibition of JNK/AP-1 activation also delays labour. In this study, to determine the potential role of JNK as a therapeutic target in PTL, we investigated the specific contribution of JNK signalling to S. Abortus LPS-induced PTL in mice. Intrauterine administration of S. Abortus LPS to pregnant mice resulted in the activation of JNK in the maternal uterus and fetal brain, upregulation of pro-inflammatory proteins COX-2, CXCL1, and CCL2, phosphorylation of cPLA2 in myometrium, and induction of PTL. Specific inhibition of JNK by co-administration of specific D-JNK inhibitory peptide (D-JNKI) delayed LPS-induced preterm delivery and reduced fetal mortality. This is associated with inhibition of myometrial cPLA2 phosphorylation and proinflammatory proteins synthesis. In addition, we report that D-JNKI inhibits the activation of JNK/JNK3 and caspase-3, which are important mediators of neural cell death in the neonatal brain. Our data demonstrate that specific inhibition of TLR4-activated JNK signalling pathways has potential as a therapeutic approach in the management of infection/inflammation-associated PTL and prevention of the associated detrimental effects to the neonatal brain. PMID:26183892

The MPS1 family of protein kinases.

PubMed

Liu, Xuedong; Winey, Mark

2012-01-01

MPS1 protein kinases are found widely, but not ubiquitously, in eukaryotes. This family of potentially dual-specific protein kinases is among several that regulate a number of steps of mitosis. The most widely conserved MPS1 kinase functions involve activities at the kinetochore in both the chromosome attachment and the spindle checkpoint. MPS1 kinases also function at centrosomes. Beyond mitosis, MPS1 kinases have been implicated in development, cytokinesis, and several different signaling pathways. Family members are identified by virtue of a conserved C-terminal kinase domain, though the N-terminal domain is quite divergent. The kinase domain of the human enzyme has been crystallized, revealing an unusual ATP-binding pocket. The activity, level, and subcellular localization of Mps1 family members are tightly regulated during cell-cycle progression. The mitotic functions of Mps1 kinases and their overexpression in some tumors have prompted the identification of Mps1 inhibitors and their active development as anticancer drugs.

The MPS1 Family of Protein Kinases

PubMed Central

Liu, Xuedong; Winey, Mark

2014-01-01

MPS1 protein kinases are found widely, but not ubiquitously, in eukaryotes. This family of potentially dual-specific protein kinases is among several that regulate a number of steps of mitosis. The most widely conserved MPS1 kinase functions involve activities at the kinetochore in both the chromosome attachment and the spindle checkpoint. MPS1 kinases also function at centrosomes. Beyond mitosis, MPS1 kinases have been implicated in development, cytokinesis, and several different signaling pathways. Family members are identified by virtue of a conserved C-terminal kinase domain, though the N-terminal domain is quite divergent. The kinase domain of the human enzyme has been crystallized, revealing an unusual ATP-binding pocket. The activity, level, and subcellular localization of Mps1 family members are tightly regulated during cell-cycle progression. The mitotic functions of Mps1 kinases and their overexpression in some tumors have prompted the identification of Mps1 inhibitors and their active development as anticancer drugs. PMID:22482908

Endoplasmic reticulum stress (ER-stress) by 2-deoxy-D-glucose (2DG) reduces cyclooxygenase-2 (COX-2) expression and N-glycosylation and induces a loss of COX-2 activity via a Src kinase-dependent pathway in rabbit articular chondrocytes.

PubMed

Yu, Seon-Mi; Kim, Song-Ja

2010-11-30

Endoplasmic reticulum (ER) stress regulates a wide range of cellular responses including apoptosis, proliferation, inflammation, and differentiation in mammalian cells. In this study, we observed the role of 2-deoxy-D-glucose (2DG) on inflammation of chondrocytes. 2DG is well known as an inducer of ER stress, via inhibition of glycolysis and glycosylation. Treatment of 2DG in chondrocytes considerably induced ER stress in a dose- and time-dependent manner, which was demonstrated by a reduction of glucose regulated protein of 94 kDa (grp94), an ER stress-inducible protein, as determined by a Western blot analysis. In addition, induction of ER stress by 2DG led to the expression of COX-2 protein with an apparent molecular mass of 66-70kDa as compared with the normally expressed 72-74 kDa protein. The suppression of ER stress with salubrinal (Salub), a selective inhibitor of eif2-alpha dephosphorylation, successfully prevented grp94 induction and efficiently recovered 2DG- modified COX-2 molecular mass and COX-2 activity might be associated with COX-2 N-glycosylation. Also, treatment of 2DG increased phosphorylation of Src in chondrocytes. The inhibition of the Src signaling pathway with PP2 (Src tyrosine kinase inhibitor) suppressed grp94 expression and restored COX-2 expression, N-glycosylation, and PGE2 production, as determined by a Western blot analysis and PGE2 assay. Taken together, our results indicate that the ER stress induced by 2DG results in a decrease of the transcription level, the molecular mass, and the activity of COX-2 in rabbit articular chondrocytes via a Src kinase-dependent pathway.

Phosphopeptide occupancy and photoaffinity cross-linking of the v-Src SH2 domain attenuates tyrosine kinase activity.

PubMed

Garcia, P; Shoelson, S E; Drew, J S; Miller, W T

1994-12-02

Phosphorylation of c-Src at carboxyl-terminal Tyr-527 suppresses tyrosine kinase activity and transforming potential, presumably by facilitating the intramolecular interaction of the C terminus of Src with its SH2 domain. In addition, it has been shown previously that occupancy of the c-Src SH2 domain with a phosphopeptide stimulates c-Src kinase catalytic activity. We have performed analogous studies with v-Src, the transforming protein from Rous sarcoma virus, which has extensive homology with c-Src. v-Src lacks an autoregulatory phosphorylation site, and its kinase domain is constitutively active. Phosphopeptides corresponding to the sequences surrounding c-Src Tyr-527 and a Tyr-Glu-Glu-Ile motif from the hamster polyoma virus middle T antigen inhibit tyrosine kinase activity of baculovirus-expressed v-Src 2- and 4-fold, respectively. To determine the mechanism of this regulation, the Tyr-527 phosphopeptide was substituted with the photoactive amino acid p-benzoylphenylalanine at the adjacent positions (N- and C-terminal) to phosphotyrosine. These peptides photoinactivate the v-Src tyrosine kinase 5-fold in a time- and concentration-dependent manner. Furthermore, the peptides cross-link an isolated Src SH2 domain with similar rates and specificity. These data indicate that occupancy of the v-Src SH2 domain induces a conformational change that is transmitted to the kinase domain and attenuates tyrosine kinase activity.

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

PubMed

Li, Xichuan; Du, Wei; Zhao, Jingwen; Zhang, Lilin; Zhu, Zhiyan; Jiang, Linghuo

2010-06-01

Rck2p is the Hog1p-MAP kinase-activated protein kinase required for the attenuation of protein synthesis in response to an osmotic challenge in Saccharomyces cerevisiae. Rck2p also regulates rapamycin sensitivity in both S. cerevisiae and Candida albicans. In this study, we demonstrate that the deletion of CaRCK2 renders C. albicans cells sensitive to, and CaRck2p translocates from the cytosol to the nucleus in response to, cell wall stresses caused by Congo red, Calcoflor White, elevated heat and zymolyase. However, the kinase activity of CaRck2p is not required for the cellular response to these cell wall stresses. Furthermore, transcripts of cell wall protein-encoding genes CaBGL2, CaHWP1 and CaXOG1 are reduced in C. albicans cells lacking CaRCK2. The deletion of CaRCK2 also reduces the in vitro filamentation of C. albicans and its virulence in a mouse model of systemic candidasis. The kinase activity of CaRck2p is required for the virulence, but not for the in vitro filamentation, in C. albicans. Therefore, Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen C. albicans.

N-terminal acetylation -an Essential Protein Modification Emerges as an Important Regulator of Stress Responses.

PubMed

Linster, Eric; Wirtz, Markus

2018-06-26

N-terminal acetylation (NTA) is a prevalent protein modification in eukaryotes. The majority of proteins is acetylated at their N-terminus in a co-translational manner by ribosome-associated N-terminal acetyltransferases (NAT). However, the recent discovery of Golgi-membrane localized NATs in metazoan, and plastid-localized NATs in plants challenged the dogma of static, co-translational imprinting of the proteome by NTA. Indeed, NTA by the cytosolic NatA is highly dynamic and under hormonal control in plants. Such active control has not been evidenced yet in other eukaryotes and might be an adaptation to the sessile lifestyle of plants forcing them to cope with diverse environmental challenges. The function of NTA for individual proteins is distinct and yet unpredictable. In yeast and humans, NTA has been shown to affect protein-protein interactions, subcellular localization, folding, aggregation, or degradation of a handful of proteins. In particular, the impact of NTA on the protein-turnover is documented by diverse examples in yeast. Consequently, NTA has recently dicovered to be a degradation signal in a distinct branch of the N-end rule pathway ubiquitin-mediated proteolysis. In this review, we summarize the current knowledge on the NAT machinery in higher plants and discuss the potential function of NTA during biotic and abiotic stresses.

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

PubMed

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

2000-02-18

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

Phosphatidylinositol 3-kinase, Cdc42, and Rac1 act downstream of Ras in integrin-dependent neurite outgrowth in N1E-115 neuroblastoma cells.

PubMed

Sarner, S; Kozma, R; Ahmed, S; Lim, L

2000-01-01

Ras and Rho family GTPases have been ascribed important roles in signalling pathways determining cellular morphology and growth. Here we investigated the roles of the GTPases Ras, Cdc42, Rac1, and Rho and that of phosphatidylinositol 3-kinase (PI 3-kinase) in the pathway leading from serum starvation to neurite outgrowth in N1E-115 neuroblastoma cells. Serum-starved cells grown on a laminin matrix exhibited integrin-dependent neurite outgrowth. Expression of dominant negative mutants of Ras, PI 3-kinase, Cdc42, or Rac1 all blocked this neurite outgrowth, while constitutively activated mutants of Ras, PI 3-kinase, or Cdc42 were each sufficient to promote outgrowth even in the presence of serum. A Ras(H40C;G12V) double mutant which binds preferentially to PI 3-kinase also promoted neurite formation. Activated Ras(G12V)-induced outgrowth required PI 3-kinase activity, but activated PI 3-kinase-induced outgrowth did not require Ras activity. Although activated Rac1 by itself did not induce neurites, neurite outgrowth induced by activated Cdc42(G12V) was Rac1 dependent. Cdc42(G12V)-induced neurites appeared to lose their normal polarization, almost doubling the average number of neurites produced by a single cell. Outgrowth induced by activated Ras or PI 3-kinase required both Cdc42 and Rac1 activity, but Cdc42(G12V)-induced outgrowth did not need Ras or PI 3-kinase activity. Active Rho(G14V) reduced outgrowth promoted by Ras(G12V). Finally, expression of dominant negative Jun N-terminal kinase or extracellular signal-regulated kinase did not inhibit outgrowth, suggesting these pathways are not essential for this process. Our results suggest a hierarchy of signalling where Ras signals through PI 3-kinase to Cdc42 and Rac1 activation (and Rho inactivation), culminating in neurite outgrowth. Thus, in the absence of serum factors, Ras may initiate cell cycle arrest and terminal differentiation in N1E-115 neuroblastoma cells.

Phosphatidylinositol 3-Kinase, Cdc42, and Rac1 Act Downstream of Ras in Integrin-Dependent Neurite Outgrowth in N1E-115 Neuroblastoma Cells

PubMed Central

Sarner, Shula; Kozma, Robert; Ahmed, Sohail; Lim, Louis

2000-01-01

Ras and Rho family GTPases have been ascribed important roles in signalling pathways determining cellular morphology and growth. Here we investigated the roles of the GTPases Ras, Cdc42, Rac1, and Rho and that of phosphatidylinositol 3-kinase (PI 3-kinase) in the pathway leading from serum starvation to neurite outgrowth in N1E-115 neuroblastoma cells. Serum-starved cells grown on a laminin matrix exhibited integrin-dependent neurite outgrowth. Expression of dominant negative mutants of Ras, PI 3-kinase, Cdc42, or Rac1 all blocked this neurite outgrowth, while constitutively activated mutants of Ras, PI 3-kinase, or Cdc42 were each sufficient to promote outgrowth even in the presence of serum. A RasH40C;G12V double mutant which binds preferentially to PI 3-kinase also promoted neurite formation. Activated RasG12V-induced outgrowth required PI 3-kinase activity, but activated PI 3-kinase-induced outgrowth did not require Ras activity. Although activated Rac1 by itself did not induce neurites, neurite outgrowth induced by activated Cdc42G12V was Rac1 dependent. Cdc42G12V-induced neurites appeared to lose their normal polarization, almost doubling the average number of neurites produced by a single cell. Outgrowth induced by activated Ras or PI 3-kinase required both Cdc42 and Rac1 activity, but Cdc42G12V-induced outgrowth did not need Ras or PI 3-kinase activity. Active RhoG14V reduced outgrowth promoted by RasG12V. Finally, expression of dominant negative Jun N-terminal kinase or extracellular signal-regulated kinase did not inhibit outgrowth, suggesting these pathways are not essential for this process. Our results suggest a hierarchy of signalling where Ras signals through PI 3-kinase to Cdc42 and Rac1 activation (and Rho inactivation), culminating in neurite outgrowth. Thus, in the absence of serum factors, Ras may initiate cell cycle arrest and terminal differentiation in N1E-115 neuroblastoma cells. PMID:10594018

Effect of oxidative stress on Rho kinase II and smooth muscle contraction in rat stomach.

PubMed

Al-Shboul, Othman; Mustafa, Ayman

2015-06-01

Recent studies have shown that both Rho kinase signaling and oxidative stress are involved in the pathogenesis of a number of human diseases, such as diabetes mellitus, hypertension, and atherosclerosis. However, very little is known about the effect of oxidative stress on the gastrointestinal (GI) smooth muscle Rho kinase pathway. The aim of the current study was to investigate the effect of oxidative stress on Rho kinase II and muscle contraction in rat stomach. The peroxynitrite donor 3-morpholinosydnonimine (SIN-1), hydrogen peroxide (H2O2), and peroxynitrite were used to induce oxidative stress. Rho kinase II expression and ACh-induced activity were measured in control and oxidant-treated cells via specifically designed enzyme-linked immunosorbent assay (ELISA) and activity assay kits, respectively. Single smooth muscle cell contraction was measured via scanning micrometry in the presence or absence of the Rho kinase blocker, Y-27632 dihydrochloride. All oxidant agents significantly increased ACh-induced Rho kinase II activity without affecting its expression level. Most important, oxidative stress induced by all three agents augmented ACh-stimulated muscle cell contraction, which was significantly inhibited by Y-27632. In conclusion, oxidative stress activates Rho kinase II and enhances contraction in rat gastric muscle, suggesting an important role in GI motility disorders associated with oxidative stress.

Distinct role of p38 and c-Jun N-terminal kinases in IL-10-dependent and IL-10-independent regulation of the costimulatory molecule B7.2 in lipopolysaccharide-stimulated human monocytic cells.

PubMed

Lim, Wilfred; Ma, Wei; Gee, Katrina; Aucoin, Susan; Nandan, Devki; Diaz-Mitoma, Francisco; Kozlowski, Maya; Kumar, Ashok

2002-02-15

The costimulatory molecule B7.2 (CD86) plays a vital role in immune activation and development of Th responses. The molecular mechanisms by which B7.2 expression is regulated are not understood. We investigated the role of mitogen-activated protein kinases (MAPK) in the regulation of B7.2 expression in LPS-stimulated human monocytic cells. LPS stimulation of human monocytes resulted in the down-regulation of B7.2 expression that could be abrogated by anti-IL-10 Abs. Furthermore, SB202190, a specific inhibitor of p38 MAPK, inhibited LPS-induced IL-10 production and reversed B7.2 down-regulation, suggesting that LPS-induced B7.2 down-regulation may be mediated, at least in part, via regulation of IL-10 production by p38 MAPK. In contrast to human promonocytic THP-1 cells that are refractory to the inhibitory effects of IL-10, LPS stimulation enhanced B7.2 expression. This IL-10-independent B7.2 induction was not influenced by specific inhibitors of either p38 or p42/44 MAPK. To ascertain the role of the c-Jun N-terminal kinase (JNK) MAPK, dexamethasone, an inhibitor of JNK activation, was used, which inhibited LPS-induced B7.2 expression. Transfection of THP-1 cells with a plasmid expressing a dominant-negative stress-activated protein/extracellular signal-regulated kinase kinase 1 significantly reduced LPS-induced B7.2 expression, thus confirming the involvement of JNK. To study the signaling events downstream of JNK activation, we show that dexamethasone did not inhibit LPS-induced NF-kappaB activation in THP-1 cells, suggesting that JNK may not be involved in NF-kappaB activation leading to B7.2 expression. Taken together, our results reveal the distinct involvement of p38 in IL-10-dependent, and JNK in IL-10-independent regulation of B7.2 expression in LPS-stimulated monocytic cells.

Site-specific Phosphorylation Protects Glycogen Synthase Kinase-3β from Calpain-mediated Truncation of Its N and C Termini*

PubMed Central

Ma, Shanshan; Liu, Shaojun; Huang, Qiaoying; Xie, Bo; Lai, Bingquan; Wang, Chong; Song, Bin; Li, Mingtao

2012-01-01

Glycogen synthase kinase-3β (GSK-3β), a key regulator of neuronal apoptosis, is inhibited by the phosphorylation of Ser-9/Ser-389 and was recently shown to be cleaved by calpain at the N terminus, leading to its subsequent activation. In this study calpain was found to cleave GSK-3β not only at the N terminus but also at the C terminus, and cleavage sites were identified at residues Thr-38–Thr-39 and Ile-384–Gln-385. Furthermore, the cleavage of GSK-3β occurred in tandem with Ser-9 dephosphorylation during cerebellar granule neuron apoptosis. Increasing Ser-9 phosphorylation of GSK-3β by inhibiting phosphatase 1/2A or pretreating with purified active Akt inhibited calpain-mediated cleavage of GSK-3β at both N and C termini, whereas non-phosphorylatable mutant GSK-3β S9A facilitated its cleavage. In contrast, Ser-389 phosphorylation selectively inhibited the cleavage of GSK-3β at the C terminus but not the N terminus. Calpain-mediated cleavage resulted in three truncated products, all of which contained an intact kinase domain: ΔN-GSK-3β (amino acids 39–420), ΔC-GSK-3β (amino acids 1–384), and ΔN/ΔC-GSK-3β (amino acids 39–384). All three truncated products showed increased kinase and pro-apoptotic activity, with ΔN/ΔC-GSK-3β being the most active form. This observation suggests that the GSK-3β C terminus acts as an autoinhibitory domain similar to the N terminus. Taken together, these findings demonstrate that calpain-mediated cleavage activates GSK-3β by removing its N- and C-terminal autoinhibitory domains and that Ser-9 phosphorylation inhibits the cleavage of GSK-3β at both termini. In contrast, Ser-389 phosphorylation inhibits only C-terminal cleavage but not N-terminal cleavage. These findings also identify a mechanism by which site-specific phosphorylation and calpain-mediated cleavage operate in concert to regulate GSK-3β activity. PMID:22496446

Site-specific phosphorylation protects glycogen synthase kinase-3β from calpain-mediated truncation of its N and C termini.

PubMed

Ma, Shanshan; Liu, Shaojun; Huang, Qiaoying; Xie, Bo; Lai, Bingquan; Wang, Chong; Song, Bin; Li, Mingtao

2012-06-29

Glycogen synthase kinase-3β (GSK-3β), a key regulator of neuronal apoptosis, is inhibited by the phosphorylation of Ser-9/Ser-389 and was recently shown to be cleaved by calpain at the N terminus, leading to its subsequent activation. In this study calpain was found to cleave GSK-3β not only at the N terminus but also at the C terminus, and cleavage sites were identified at residues Thr-38-Thr-39 and Ile-384-Gln-385. Furthermore, the cleavage of GSK-3β occurred in tandem with Ser-9 dephosphorylation during cerebellar granule neuron apoptosis. Increasing Ser-9 phosphorylation of GSK-3β by inhibiting phosphatase 1/2A or pretreating with purified active Akt inhibited calpain-mediated cleavage of GSK-3β at both N and C termini, whereas non-phosphorylatable mutant GSK-3β S9A facilitated its cleavage. In contrast, Ser-389 phosphorylation selectively inhibited the cleavage of GSK-3β at the C terminus but not the N terminus. Calpain-mediated cleavage resulted in three truncated products, all of which contained an intact kinase domain: ΔN-GSK-3β (amino acids 39-420), ΔC-GSK-3β (amino acids 1-384), and ΔN/ΔC-GSK-3β (amino acids 39-384). All three truncated products showed increased kinase and pro-apoptotic activity, with ΔN/ΔC-GSK-3β being the most active form. This observation suggests that the GSK-3β C terminus acts as an autoinhibitory domain similar to the N terminus. Taken together, these findings demonstrate that calpain-mediated cleavage activates GSK-3β by removing its N- and C-terminal autoinhibitory domains and that Ser-9 phosphorylation inhibits the cleavage of GSK-3β at both termini. In contrast, Ser-389 phosphorylation inhibits only C-terminal cleavage but not N-terminal cleavage. These findings also identify a mechanism by which site-specific phosphorylation and calpain-mediated cleavage operate in concert to regulate GSK-3β activity.

The effect of carboxydextran-coated superparamagnetic iron oxide nanoparticles on c-Jun N-terminal kinase-mediated apoptosis in human macrophages.

PubMed

Lunov, Oleg; Syrovets, Tatiana; Büchele, Berthold; Jiang, Xiue; Röcker, Carlheinz; Tron, Kyrylo; Nienhaus, G Ulrich; Walther, Paul; Mailänder, Volker; Landfester, Katharina; Simmet, Thomas

2010-07-01

Superparamagnetic iron oxide nanoparticles are frequently used for cell labeling or as diagnostic contrast media, yet studies analyzing their effects on immune cells remain scarce. Here we investigated how nanosized carboxydextran-coated superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO) might affect human macrophages. Within 1 h, both SPIO and USPIO were rapidly taken up by macrophages. Confocal microscopy revealed that after 24 h the particles were almost exclusively localized within the lysosomal compartment. Continued cultivation of the macrophages for several days was associated with apoptosis induction caused by a long-lasting activation of the c-Jun N-terminal kinase (JNK) pathway. JNK activation was due to significantly elevated levels of reactive oxygen species, whereas no TNF-alpha was produced by the macrophages treated with nanoparticles. Compared to SPIO, USPIO induced more pronounced biochemical alterations and cytotoxicity, which could be antagonized by the JNK inhibitor V. Alternatively, treatment of macrophages with Trolox or N-acetyl-L-cysteine, two functionally different scavengers of reactive oxygen species, abolished both the JNK activation and the subsequent cytotoxic effects. These data indicate that nanosized superparamagnetic iron oxide-based contrast media exert cytotoxicity in human macrophages that can be functionally antagonized with radical scavengers. Copyright 2010 Elsevier Ltd. All rights reserved.

Effect of a glutathione S-transferase inhibitor on oxidative stress and ischemia-reperfusion-induced apoptotic signalling of cultured cardiomyocytes

PubMed Central

Röth, E; Marczin, N; Balatonyi, B; Ghosh, S; Kovács, V; Alotti, N; Borsiczky, B; Gasz, B

2011-01-01

Oxidative stress and ischemia-reperfusion (I/R) injury are crucial in the pathogenesis of cardiovascular diseases. The antioxidant glutathione S-transferase (GST) is responsible for the high-capacity metabolic inactivation of electrophilic compounds and toxic substrates. The main objective of the present study was to examine the effect of GST inhibition (with the administration of ethacrynic acid [EA]) on the viability and apoptosis of cardiomyocytes when these cells are exposed to various stress components of I/R and mitogen-activated protein kinase (c-Jun N-terminal kinase, p38 and extracellular signal-regulated kinase [ERK]) inhibitors. The primary culture of neonatal rat cardiomyocytes was divided into six experimental groups: control group of cells (group 1), cells exposed to H2O2 (group 2), I/R (group 3), I/R and EA (group 4), H2O2 coupled with EA (group 5), and EA alone (group 6). The viability of cardiomyocytes was determined using a colorimetric MTT assay. The apoptosis ratio was evaluated via fluorescein isothiocyanate-labelled annexin V and propidium iodide staining. c-Jun N-terminal kinase, p38, Akt/protein kinase B and ERK/p42-p44 transcription factors were monitored with flow cytometry. c-Jun N-terminal kinase activation increased due to GST inhibition during I/R. EA administration led to a significant increase in p38 activation following both H2O2 treatment and I/R. ERK phosphorylation increased when GST was exposed to I/R. A pronounced decrease in Akt phosphorylation was observed when cells were cotreated with EA and H2O2. GST plays an important role as a regulator of mitogen-activated protein kinase pathways in I/R injury. PMID:22065940

SAM domain-dependent activity of PfTKL3, an essential tyrosine kinase-like kinase of the human malaria parasite Plasmodium falciparum.

PubMed

Abdi, Abdirahman; Eschenlauer, Sylvain; Reininger, Luc; Doerig, Christian

2010-10-01

Over the last decade, several protein kinases inhibitors have reached the market for cancer chemotherapy. The kinomes of pathogens represent potentially attractive targets in infectious diseases. The functions of the majority of protein kinases of Plasmodium falciparum, the parasitic protist responsible for the most virulent form of human malaria, remain unknown. Here we present a thorough characterisation of PfTKL3 (PF13_0258), an enzyme that belongs to the tyrosine kinase-like kinase (TKL) group. We demonstrate by reverse genetics that PfTKL3 is essential for asexual parasite proliferation in human erythrocytes. PfTKL3 is expressed in both asexual and gametocytes stages, and in the latter the protein co-localises with cytoskeleton microtubules. Recombinant PfTKL3 displays in vitro autophosphorylation activity and is able to phosphorylate exogenous substrates, and both activities are dramatically dependent on the presence of an N-terminal "sterile alpha-motif" domain. This study identifies PfTKL3 as a validated drug target amenable to high-throughput screening.

Mps1 Phosphorylates Its N-Terminal Extension to Relieve Autoinhibition and Activate the Spindle Assembly Checkpoint.

PubMed

Combes, Guillaume; Barysz, Helena; Garand, Chantal; Gama Braga, Luciano; Alharbi, Ibrahim; Thebault, Philippe; Murakami, Luc; Bryne, Dominic P; Stankovic, Stasa; Eyers, Patrick A; Bolanos-Garcia, Victor M; Earnshaw, William C; Maciejowski, John; Jallepalli, Prasad V; Elowe, Sabine

2018-03-19

Monopolar spindle 1 (Mps1) is a conserved apical kinase in the spindle assembly checkpoint (SAC) that ensures accurate segregation of chromosomes during mitosis. Mps1 undergoes extensive auto- and transphosphorylation, but the regulatory and functional consequences of these modifications remain unclear. Recent findings highlight the importance of intermolecular interactions between the N-terminal extension (NTE) of Mps1 and the Hec1 subunit of the NDC80 complex, which control Mps1 localization at kinetochores and activation of the SAC. Whether the NTE regulates other mitotic functions of Mps1 remains unknown. Here, we report that phosphorylation within the NTE contributes to Mps1 activation through relief of catalytic autoinhibition that is mediated by the NTE itself. Moreover, we find that this regulatory NTE function is independent of its role in Mps1 kinetochore recruitment. We demonstrate that the NTE autoinhibitory mechanism impinges most strongly on Mps1-dependent SAC functions and propose that Mps1 activation likely occurs sequentially through dimerization of a "prone-to-autophosphorylate" Mps1 conformer followed by autophosphorylation of the NTE prior to maximal kinase activation segment trans-autophosphorylation. Our observations underline the importance of autoregulated Mps1 activity in generation and maintenance of a robust SAC in human cells. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Unpredictable chronic mild stress induces anxiety and depression-like behaviors and inactivates AMP-activated protein kinase in mice.

PubMed

Zhu, Shenghua; Wang, Junhui; Zhang, Yanbo; Li, Victor; Kong, Jiming; He, Jue; Li, Xin-Min

2014-08-12

The unpredictable chronic mild stress (UCMS) model was developed based upon the stress-diathesis hypothesis of depression. Most effects of UCMS can be reversed by antidepressants, demonstrating a strong predictive validity of this model for depression. However, the mechanisms underlying the effects induced by UCMS remain incompletely understood. Increasing evidence has shown that AMP-activated protein kinase (AMPK) regulates intracellular energy metabolism and is especially important for neurons because neurons are known to have small energy reserves. Abnormalities in the AMPK pathway disturb normal brain functions and synaptic integrity. In the present study, we first investigated the effects of UCMS on a battery of different tests measuring anxiety and depression-like behaviors in female C57BL/6N mice after 4 weeks of UCMS exposure. Stressed mice showed suppressed body weight gain, heightened anxiety, and increased immobility in the forced swim and tail suspension tests. These results are representative of some of the core symptoms of depression. Simultaneously, we observed decrease of synaptic proteins in the cortex of mice subjected to UCMS, which is associated with decreased levels of phosphorylated AMP-activated protein kinase α (AMPKα) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase). Our findings suggest that AMPKα inactivation might be a mechanism by which UCMS causes anxiety/depression-like behaviors in mice. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterization of Runella slithyformis HD-Pnk, a bifunctional DNA/RNA end-healing enzyme composed of an N-terminal 2',3' -phosphoesterase HD domain and a C-terminal 5' -OH polynucleotide kinase domain.

PubMed

Munir, Annum; Shuman, Stewart

2016-11-28

5' and 3' end healing are key steps in nucleic acid break repair in which 5' -OH ends are phosphorylated by a polynucleotide kinase and 3' -PO 4 or 2',3' -cyclic-PO 4 ends are hydrolyzed by a phosphoesterase to generate the 5' -PO 4 and 3' -OH termini required for sealing by classic polynucleotide ligases. End healing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2',3' -phosphoesterase HD domain and a C-terminal 5' -OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5' -OH polynucleotides (9-mers or longer) in the presence of magnesium and any NTP donor. HD-Pnk dephosphorylates RNA 2',3' -cyclic phosphate, RNA 3' -phosphate, RNA 2' -phosphate, and DNA 3' -phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper or cobalt. HD-Pnkp homologs are present in genera from eleven bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. The present study provides insights to the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnkp as the exemplar of a novel clade of dual 5' and 3' end-healing enzymes that phosphorylate 5' -OH termini and dephosphorylate 2',3' -cyclic-PO 4 , 3' -PO 4 , and 2' -PO 4 ends. The distinctive feature of HD-Pnk is its domain composition: a fusion of an N-terminal HD phosphohydrolase module to a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, domain order, and similar polypeptide size are distributed widely among genera from eleven bacterial phyla. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii

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

Qiu, Wei; Wernimont, Amy; Tang, Keliang

2009-09-29

Serine/threonine kinases secreted from rhoptry organelles constitute important virulence factors of Toxoplasma gondii. Rhoptry kinases are highly divergent and their structures and regulatory mechanism are hitherto unknown. Here, we report the X-ray crystal structures of two related pseudokinases named ROP2 and ROP8, which differ primarily in their substrate-binding site. ROP kinases contain a typical bilobate kinase fold and a novel N-terminal extension that both stabilizes the N-lobe and provides a unique means of regulation. Although ROP2 and ROP8 were catalytically inactive, they provided a template for homology modelling of the active kinase ROP18, a major virulence determinant of T. gondii.more » Autophosphorylation of key residues in the N-terminal extension resulted in ROP18 activation, which in turn phosphorylated ROP2 and ROP8. Mutagenesis and mass spectrometry experiments revealed that ROP18 was maximally activated when this phosphorylated N-terminus relieved autoinhibition resulting from extension of aliphatic side chains into the ATP-binding pocket. This novel means of regulation governs ROP kinases implicated in parasite virulence.« less

Protective effect of black garlic extracts on tert-Butyl hydroperoxide-induced injury in hepatocytes via a c-Jun N-terminal kinase-dependent mechanism

PubMed Central

Lee, Ko-Chao; Teng, Chih-Chuan; Shen, Chien-Heng; Huang, Wen-Shih; Lu, Chien-Chang; Kuo, Hsing-Chun; Tung, Shui-Yi

2018-01-01

Black garlic has been reported to show multiple bioactivities against the development of different diseases. In the present study, the hepatoprotective effect of black garlic on injured liver cells was investigated. Rat clone-9 hepatocytes were used for all experiments; tert-Butyl hydroperoxide (tBHP) was used to induce injury of rat clone-9 hepatocytes. The contents of malondialdehyde (MDA) and glutathione (GSH); anti-oxidative enzyme activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx); and mRNA expression levels of interleukin (IL)-6 and IL-8 in rat clone-9 hepatocytes were determined to evaluate the level of cell damage. Black garlic extracts were demonstrated to significantly attenuate tBHP-induced cell death of rat clone-9 hepatocytes (P<0.05). Pretreatment with black garlic extracts antagonized GSH depletion, tBHP-increased MDA accumulation and the mRNA expression level of IL-6/IL-8, and tBHP-decreased antioxidative enzyme activities (all P<0.05). Moreover, the present study revealed that c-Jun N-terminal kinase signaling regulated black garlic-inhibited tBHP effects in rat clone-9 hepatocytes. Our findings demonstrate that black garlic has the hepatoprotective potential to block tBHP-damaged effects on cell death, lipid peroxidation, oxidative stress, and inflammation in rat clone-9 hepatocytes. Thus, the present study indicates that black garlic may be an excellent natural candidate in the development of adjuvant therapy and healthy foods for liver protection. PMID:29456651

The Green Tea Component (-)-Epigallocatechin-3-Gallate Sensitizes Primary Endothelial Cells to Arsenite-Induced Apoptosis by Decreasing c-Jun N-Terminal Kinase-Mediated Catalase Activity

PubMed Central

Lee, Hyeon-Ju; Byun, Catherine Jeonghae; Park, Jung-Hyun; Park, Jae Hoon; Cho, Ho-Seong; Cho, Sung-Jin; Jo, Sangmee Ahn; Jo, Inho

2015-01-01

The green tea component (-)-epigallocatechin-3-gallate (EGCG) has been shown to sensitize many different types of cancer cells to anticancer drug-induced apoptosis, although it protects against non-cancerous primary cells against toxicity from certain conditions such as exposure to arsenic (As) or ultraviolet irradiation. Here, we found that EGCG promotes As-induced toxicity of primary-cultured bovine aortic endothelial cells (BAEC) at doses in which treatment with each chemical alone had no such effect. Increased cell toxicity was accompanied by an increased condensed chromatin pattern and fragmented nuclei, cleaved poly(ADP-ribose) polymerase (PARP), activity of the pro-apoptotic enzymes caspases 3, 8 and 9, and Bax translocation into mitochondria, suggesting the involvement of an apoptotic signaling pathway. Fluorescence activated cell sorting analysis revealed that compared with EGCG or As alone, combined EGCG and As (EGCG/As) treatment significantly induced production of reactive oxygen species (ROS), which was accompanied by decreased catalase activity and increased lipid peroxidation. Pretreatment with N-acetyl-L-cysteine or catalase reversed EGCG/As-induced caspase activation and EC toxicity. EGCG/As also increased the phosphorylation of c-Jun N-terminal kinase (JNK), which was not reversed by catalase. However, pretreatment with the JNK inhibitor SP600125 reversed all of the observed effects of EGCG/As, suggesting that JNK may be the most upstream protein examined in this study. Finally, we also found that all the observed effects by EGCG/As are true for other types of EC tested. In conclusion, this is firstly to show that EGCG sensitizes non-cancerous EC to As-induced toxicity through ROS-mediated apoptosis, which was attributed at least in part to a JNK-activated decrease in catalase activity. PMID:26375285

The Green Tea Component (-)-Epigallocatechin-3-Gallate Sensitizes Primary Endothelial Cells to Arsenite-Induced Apoptosis by Decreasing c-Jun N-Terminal Kinase-Mediated Catalase Activity.

PubMed

Kim, Jee-Youn; Choi, Ji-Young; Lee, Hyeon-Ju; Byun, Catherine Jeonghae; Park, Jung-Hyun; Park, Jae Hoon; Cho, Ho-Seong; Cho, Sung-Jin; Jo, Sangmee Ahn; Jo, Inho

2015-01-01

The green tea component (-)-epigallocatechin-3-gallate (EGCG) has been shown to sensitize many different types of cancer cells to anticancer drug-induced apoptosis, although it protects against non-cancerous primary cells against toxicity from certain conditions such as exposure to arsenic (As) or ultraviolet irradiation. Here, we found that EGCG promotes As-induced toxicity of primary-cultured bovine aortic endothelial cells (BAEC) at doses in which treatment with each chemical alone had no such effect. Increased cell toxicity was accompanied by an increased condensed chromatin pattern and fragmented nuclei, cleaved poly(ADP-ribose) polymerase (PARP), activity of the pro-apoptotic enzymes caspases 3, 8 and 9, and Bax translocation into mitochondria, suggesting the involvement of an apoptotic signaling pathway. Fluorescence activated cell sorting analysis revealed that compared with EGCG or As alone, combined EGCG and As (EGCG/As) treatment significantly induced production of reactive oxygen species (ROS), which was accompanied by decreased catalase activity and increased lipid peroxidation. Pretreatment with N-acetyl-L-cysteine or catalase reversed EGCG/As-induced caspase activation and EC toxicity. EGCG/As also increased the phosphorylation of c-Jun N-terminal kinase (JNK), which was not reversed by catalase. However, pretreatment with the JNK inhibitor SP600125 reversed all of the observed effects of EGCG/As, suggesting that JNK may be the most upstream protein examined in this study. Finally, we also found that all the observed effects by EGCG/As are true for other types of EC tested. In conclusion, this is firstly to show that EGCG sensitizes non-cancerous EC to As-induced toxicity through ROS-mediated apoptosis, which was attributed at least in part to a JNK-activated decrease in catalase activity.

c-Jun N-terminal kinase 3 (JNK3) Mediates Paraquat- and Rotenone-Induced Dopaminergic Neuron Death

PubMed Central

Choi, Won Seok; Abel, Glen; Klintworth, Heather; Flavell, Richard A.; Xia, Zhengui

2011-01-01

Mechanistic studies underlying dopaminergic neuron death may identify new drug targets for the treatment of Parkinson disease (PD). Epidemiological studies have linked pesticide exposure to increased risk for sporadic PD. Here, we investigated the role of c-Jun N-terminal kinase 3 (JNK3), a neural-specific JNK isoform, in dopaminergic neuron death induced by the pesticides rotenone and paraquat. The role of JNK3 was evaluated using RNA silencing and gene deletion to block JNK3 signaling. Using an antibody that recognizes all isoforms of activated JNKs, we found that paraquat and rotenone stimulate JNK phosphorylation in primary cultured dopaminergic neurons. In cultured neurons transfected with Jnk3-specific siRNA and in neurons from Jnk3−/− mice, JNK phosphorylation was nearly abolished, suggesting that JNK3 is the main JNK isoform activated in dopaminergic neurons by these pesticides. Paraquat- and rotenone-induced death of dopaminergic neurons was also significantly reduced by Jnk3 siRNA or Jnk3 gene deletion and deletion of the Jnk3 gene completely attenuated paraquat-induced dopaminergic neuron death and motor-deficits in vivo. Our data identify JNK3 as a common and critical mediator of dopaminergic neuron death induced by paraquat and rotenone, suggesting that it is a potential drug target for PD treatment. PMID:20418776

Insufficiency of pro-heparin-binding epidermal growth factor-like growth factor shedding enhances hypoxic cell death in H9c2 cardiomyoblasts via the activation of caspase-3 and c-Jun N-terminal kinase.

PubMed

Uetani, Teruyoshi; Nakayama, Hironao; Okayama, Hideki; Okura, Takafumi; Higaki, Jitsuo; Inoue, Hirofumi; Higashiyama, Shigeki

2009-05-01

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cardiogenic and cardiohypertrophic growth factor. ProHB-EGF, a product of the Hb-egf gene and the precursor of HB-EGF, is anchored to the plasma membrane. Its ectodomain region is shed by a disintegrin and metalloproteases (ADAMs) when activated by various stimulations. It has been reported that an uncleavable mutant of Hb-egf, uc-Hb-egf, produces uc-proHB-EGF, which is not cleaved by ADAMs and causes dilation of the heart in knock-in mice. This suggests that the shedding of proHB-EGF is essential for the development and survival of cardiomyocytes: however, the molecular mechanism involved has remained unclear. In this study, we investigated the relationship between uc-proHB-EGF expression and cardiomyocyte survival. Human uc-proHB-EGF was adenovirally introduced into the rat cardiomyoblast cell line H9c2, and the cells were cultured under normoxic and hypoxic conditions. Uc-proHB-EGF-expressing H9c2 cells underwent apoptosis under normoxic conditions, which distinctly increased under hypoxic conditions. Furthermore, we observed an increased Caspase-3 activity, reactive oxygen species accumulation, and an increased c-Jun N-terminal kinase (JNK) activity in the uc-proHB-EGF-expressing H9c2 cells. Treatment of the uc-proHB-EGF transfectants with inhibitors of Caspase-3, reactive oxygen species, and JNK, namely, Z-VAD-fmk, N-acetylcysteine, and SP600125, respectively, significantly reduced hypoxic cell death. These data indicate that insufficiency of proHB-EGF shedding under hypoxic stress leads to cardiomyocyte apoptosis via Caspase-3- and JNK-dependent pathways.

Structure-activity relationships of phenothiazines and related drugs for inhibition of protein kinase C.

PubMed

Aftab, D T; Ballas, L M; Loomis, C R; Hait, W N

1991-11-01

Phenothiazines are known to inhibit the activity of protein kinase C. To identify structural features that determine inhibitory activity against the enzyme, we utilized a semiautomated assay [Anal. Biochem. 187:84-88 (1990)] to compare the potency of greater than 50 phenothiazines and related compounds. Potency was decreased by trifluoro substitution at position 2 on the phenothiazine nucleus and increased by quinoid structures on the nucleus. An alkyl bridge of at least three carbons connecting the terminal amine to the nucleus was required for activity. Primary amines and unsubstituted piperazines were the most potent amino side chains. We selected 7,8-dihydroxychlorpromazine (DHCP) (IC50 = 8.3 microM) and 2-chloro-9-(3-[1-piperazinyl]propylidene)thioxanthene (N751) (IC50 = 14 microM) for further study because of their potency and distinct structural features. Under standard (vesicle) assay conditions, DHCP was noncompetitive with respect to phosphatidylserine and a mixed-type inhibitor with respect to ATP. N751 was competitive with respect to phosphatidylserine and noncompetitive with respect to ATP. Using the mixed micelle assay, DHCP was a competitive inhibitor with respect to both phosphatidylserine and ATP. DHCP was selective for protein kinase C compared with cAMP-dependent protein kinase, calmodulin-dependent protein kinase type II, and casein kinase. N751 was more potent against protein kinase C compared with cAMP-dependent protein kinase and casein kinase but less potent against protein kinase C compared with calmodulin-dependent protein kinase type II. DHCP was analyzed for its ability to inhibit different isoenzymes of protein kinase C, and no significant isozyme selectivity was detected. These data provide important information for the rational design of more potent and selective inhibitors of protein kinase C.

Gallic Acid Induces a Reactive Oxygen Species-Provoked c-Jun NH2-Terminal Kinase-Dependent Apoptosis in Lung Fibroblasts

PubMed Central

Chen, Chiu-Yuan; Chen, Kun-Chieh; Yang, Tsung-Ying; Liu, Hsiang-Chun; Hsu, Shih-Lan

2013-01-01

Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by fibroblasts proliferation and extracellular matrix accumulation. Induction of fibroblast apoptosis therefore plays a crucial role in the resolution of this disease. Gallic acid (3,4,5-trihydroxybenzoic acid), a common botanic phenolic compound, has been reported to induce apoptosis in tumor cell lines and renal fibroblasts. The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in lung fibroblasts apoptosis induced by gallic acid. We found that treatment with gallic acid resulted in activation of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB, Akt), but not p38MAPK, in mouse lung fibroblasts. Inhibition of JNK using pharmacologic inhibitor (SP600125) and genetic knockdown (JNK specific siRNA) significantly inhibited p53 accumulation, reduced PUMA and Fas expression, and abolished apoptosis induced by gallic acid. Moreover, treatment with antioxidants (vitamin C, N-acetyl cysteine, and catalase) effectively diminished gallic acid-induced hydrogen peroxide production, JNK and p53 activation, and cell death. These observations imply that gallic acid-mediated hydrogen peroxide formation acts as an initiator of JNK signaling pathways, leading to p53 activation and apoptosis in mouse lung fibroblasts. PMID:23533505

Particulate air pollution induces arrhythmia via oxidative stress and calcium calmodulin kinase II activation

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

Kim, Jin-Bae; Kim, Changsoo; Choi, Eunmi

2012-02-15

Ambient particulate matter (PM) can increase the incidence of arrhythmia. However, the arrhythmogenic mechanism of PM is poorly understood. This study investigated the arrhythmogenic mechanism of PM. In Sprague–Dawley rats, QT interval was increased from 115.0 ± 14.0 to 142.1 ± 18.4 ms (p = 0.02) after endotracheal exposure of DEP (200 μg/ml for 30 min, n = 5). Ventricular premature contractions were more frequently observed after DEP exposure (100%) than baseline (20%, p = 0.04). These effects were prevented by pretreatment of N-acetylcysteine (NAC, 5 mmol/L, n = 3). In 12 Langendorff-perfused rat hearts, DEP infusion of 12.5 μg/mlmore » for 20 min prolonged action potential duration (APD) at only left ventricular base increasing apicobasal repolarization gradients. Spontaneous early afterdepolarization (EAD) and ventricular tachycardia (VT) were observed in 8 (67%) and 6 (50%) hearts, respectively, versus no spontaneous triggered activity or VT in any hearts before DEP infusion. DEP-induced APD prolongation, EAD and VT were successfully prevented with NAC (5 mmol/L, n = 5), nifedipine (10 μmol/L, n = 5), and active Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) blockade, KN 93 (1 μmol/L, n = 5), but not by thapsigargin (200 nmol/L) plus ryanodine (10 μmol/L, n = 5) and inactive CaMKII blockade, KN 92 (1 μmol/L, n = 5). In neonatal rat cardiomyocytes, DEP provoked ROS generation in dose dependant manner. DEP (12.5 μg/ml) induced apoptosis, and this effect was prevented by NAC and KN 93. Thus, this study shows that in vivo and vitro exposure of PM induced APD prolongation, EAD and ventricular arrhythmia. These effects might be caused by oxidative stress and CaMKII activation. -- Highlights: ► The ambient PM consistently prolonged repolarization. ► The ambient PM induced triggered activity and ventricular arrhythmia. ► These effects were prevented by antioxidants, I{sub CaL} blockade and CaMKII blockade. ► The ambient PM can induce

Human T-Cell Leukemia Virus Type 1 Tax Induction of NF-κB Involves Activation of the IκB Kinase α (IKKα) and IKKβ Cellular Kinases

PubMed Central

Geleziunas, Romas; Ferrell, Sharon; Lin, Xin; Mu, Yajun; Cunningham, Emmett T.; Grant, Mark; Connelly, Margery A.; Hambor, John E.; Marcu, Kenneth B.; Greene, Warner C.

1998-01-01

Tax corresponds to a 40-kDa transforming protein from the pathogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) that activates nuclear expression of the NF-κB/Rel family of transcription factors by an unknown mechanism. Tax expression promotes N-terminal phosphorylation and degradation of IκBα, a principal cytoplasmic inhibitor of NF-κB. Our studies now demonstrate that HTLV-1 Tax activates the recently identified cellular kinases IκB kinase α (IKKα) and IKKβ, which normally phosphorylate IκBα on both of its N-terminal regulatory serines in response to tumor necrosis factor alpha (TNF-α) and interleukin-1 (IL-1) stimulation. In contrast, a mutant of Tax termed M22, which does not induce NF-κB, fails to activate either IKKα or IKKβ. Furthermore, endogenous IKK enzymatic activity was significantly elevated in HTLV-1-infected and Tax-expressing T-cell lines. Transfection of kinase-deficient mutants of IKKα and IKKβ into either human Jurkat T or 293 cells also inhibits NF-κB-dependent reporter gene expression induced by Tax. Similarly, a kinase-deficient mutant of NIK (NF-κB-inducing kinase), which represents an upstream kinase in the TNF-α and IL-1 signaling pathways leading to IKKα and IKKβ activation, blocks Tax induction of NF-κB. However, plasma membrane-proximal elements in these proinflammatory cytokine pathways are apparently not involved since dominant negative mutants of the TRAF2 and TRAF6 adaptors, which effectively block signaling through the cytoplasmic tails of the TNF-α and IL-1 receptors, respectively, do not inhibit Tax induction of NF-κB. Together, these studies demonstrate that HTLV-1 Tax exploits a distal part of the proinflammatory cytokine signaling cascade leading to induction of NF-κB. The pathological alteration of this cytokine pathway leading to NF-κB activation by Tax may play a central role in HTLV-1-mediated transformation of human T cells, clinically manifested as the adult T-cell leukemia. PMID

Seasonal variations of cellular stress response in the heart and gastrocnemius muscle of the water frog (Pelophylax ridibundus).

PubMed

Feidantsis, Konstantinos; Anestis, Andreas; Vasara, Eleni; Kyriakopoulou-Sklavounou, Pasqualina; Michaelidis, Basile

2012-08-01

The present study aimed to investigate the seasonal cellular stress response in the heart and the gastrocnemius muscle of the amphibian Pelophylax ridibundus (former name Rana ridibunda) during an 8 month acclimatization period in the field. Processes studied included heat shock protein expression and protein kinase activation. The cellular stress response was addressed through the expression of Hsp70 and Hsp90 and the phosphorylation of stress-activated protein kinases and particularly p38 mitogen-activated protein kinase (p38 MAPK), the extracellular signal-regulated kinases (ERK-1/2) and c-Jun N-terminal kinases (JNK1/2/3). Due to a general metabolic depression during winter hibernation, the induction of Hsp70 and Hsp90 and the phosphorylation of p38 MAPK, JNKs and ERKs are retained at low levels of expression in the examined tissues of P. ridibundus. Recovery from hibernation induces increased levels of the specific proteins, probably providing stamina to the animals during their arousal. Copyright © 2012 Elsevier Inc. All rights reserved.

The β subunit of yeast AMP-activated protein kinase directs substrate specificity in response to alkaline stress

PubMed Central

Chandrashekarappa, Dakshayini G.; McCartney, Rhonda R.; O’Donnell, Allyson F.; Schmidt, Martin C.

2016-01-01

Saccharomyces cerevisiae express three isoforms of Snf1 kinase that differ by which β subunit is present, Gal83, Sip1 or Sip2. Here we investigate the abundance, activation, localization and signaling specificity of the three Snf1 isoforms. The relative abundance of these isoforms was assessed by quantitative immunoblotting using two different protein extraction methods and by fluorescence microscopy. The Gal83 containing isoform is the most abundant in all assays while the abundance of the Sip1 and Sip2 isoforms is typically underestimated especially in glass-bead extractions. Earlier studies to assess Snf1 isoform function utilized gene deletions as a means to inactivate specific isoforms. Here we use point mutations in Gal83 and Sip2 and a 17 amino acid C-terminal truncation of Sip1 to inactivate specific isoforms without affecting their abundance or association with the other subunits. The effect of low glucose and alkaline stresses was examined for two Snf1 phosphorylation substrates, the Mig1 and Mig2 proteins. Any of the three isoforms was capable of phosphorylating Mig1 in response to glucose stress. In contrast, the Gal83 isoform of Snf 1 was both necessary and sufficient for the phosphorylation of the Mig2 protein in response to alkaline stress. Alkaline stress led to the activation of all three isoforms yet only the Gal83 isoform translocates to the nucleus and phosphorylates Mig2. Deletion of the SAK1 gene blocked nuclear translocation of Gal83 and signaling to Mig2. These data strongly support the idea that Snf1 signaling specificity is mediated by localization of the different Snf1 isoforms. PMID:27592031

Structure of the intact ATM/Tel1 kinase

NASA Astrophysics Data System (ADS)

Wang, Xuejuan; Chu, Huanyu; Lv, Mengjuan; Zhang, Zhihui; Qiu, Shuwan; Liu, Haiyan; Shen, Xuetong; Wang, Weiwu; Cai, Gang

2016-05-01

The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents.

The Haemophilus ducreyi LspA1 protein inhibits phagocytosis by using a new mechanism involving activation of C-terminal Src kinase.

PubMed

Dodd, Dana A; Worth, Randall G; Rosen, Michael K; Grinstein, Sergio; van Oers, Nicolai S C; Hansen, Eric J

2014-05-20

Haemophilus ducreyi causes chancroid, a sexually transmitted infection. A primary means by which this pathogen causes disease involves eluding phagocytosis; however, the molecular basis for this escape mechanism has been poorly understood. Here, we report that the LspA virulence factors of H. ducreyi inhibit phagocytosis by stimulating the catalytic activity of C-terminal Src kinase (Csk), which itself inhibits Src family protein tyrosine kinases (SFKs) that promote phagocytosis. Inhibitory activity could be localized to a 37-kDa domain (designated YL2) of the 456-kDa LspA1 protein. The YL2 domain impaired ingestion of IgG-opsonized targets and decreased levels of active SFKs when expressed in mammalian cells. YL2 contains tyrosine residues in two EPIYG motifs that are phosphorylated in mammalian cells. These tyrosine residues were essential for YL2-based inhibition of phagocytosis. Csk was identified as the predominant mammalian protein interacting with YL2, and a dominant-negative Csk rescued phagocytosis in the presence of YL2. Purified Csk phosphorylated the tyrosines in the YL2 EPIYG motifs. Phosphorylated YL2 increased Csk catalytic activity, resulting in positive feedback, such that YL2 can be phosphorylated by the same kinase that it activates. Finally, we found that the Helicobacter pylori CagA protein also inhibited phagocytosis in a Csk-dependent manner, raising the possibility that this may be a general mechanism among diverse bacteria. Harnessing Csk to subvert the Fcγ receptor (FcγR)-mediated phagocytic pathway represents a new bacterial mechanism for circumventing a crucial component of the innate immune response and may potentially affect other SFK-involved cellular pathways. Phagocytosis is a critical component of the immune system that enables pathogens to be contained and cleared. A number of bacterial pathogens have developed specific strategies to either physically evade phagocytosis or block the intracellular signaling required for

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

PubMed Central

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

1997-01-01

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

OXIDATIVE STRESS PARTICIPATES IN ACUTE LUNG INJURY AND ACTIVATION OF MITOGEN ACTIVATED PROTEIN KINASES (MAPK) FOLLOWING AIR POLLUTION PARTICLE EXPOSURE (PM)

EPA Science Inventory

OXIDATIVE STRESS PARTICIPATES IN ACUTE LUNG INJURY AND ACTIVATION OF MITOGEN ACTIVATED PROTEIN KINASES (MAPK) FOLLOWING AIR POLLUTION PARTICLE EXPOSURE (PM). E S Roberts1, R Jaskot2, J Richards2, and K L Dreher2. 1College of Veterinary Medicine, NC State University, Raleigh, NC a...

AMP Kinase Activation Alters Oxidant-Induced Stress Granule Assembly by Modulating Cell Signaling and Microtubule Organization.

PubMed

Mahboubi, Hicham; Koromilas, Antonis E; Stochaj, Ursula

2016-10-01

Eukaryotic cells assemble stress granules (SGs) when translation initiation is inhibited. Different cell signaling pathways regulate SG production. Particularly relevant to this process is 5'-AMP-activated protein kinase (AMPK), which functions as a stress sensor and is transiently activated by adverse physiologic conditions. Here, we dissected the role of AMPK for oxidant-induced SG formation. Our studies identified multiple steps of de novo SG assembly that are controlled by the kinase. Single-cell analyses demonstrated that pharmacological AMPK activation prior to stress exposure changed SG properties, because the granules became more abundant and smaller in size. These altered SG characteristics correlated with specific changes in cell survival, cell signaling, cytoskeletal organization, and the abundance of translation initiation factors. Specifically, AMPK activation increased stress-induced eukaryotic initiation factor (eIF) 2α phosphorylation and reduced the concentration of eIF4F complex subunits eIF4G and eIF4E. At the same time, the abundance of histone deacetylase 6 (HDAC6) was diminished. This loss of HDAC6 was accompanied by increased acetylation of α-tubulin on Lys40. Pharmacological studies further confirmed this novel AMPK-HDAC6 interplay and its importance for SG biology. Taken together, we provide mechanistic insights into the regulation of SG formation. We propose that AMPK activation stimulates oxidant-induced SG formation but limits their fusion into larger granules. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Design and synthesis of the first generation of novel potent, selective, and in vivo active (benzothiazol-2-yl)acetonitrile inhibitors of the c-Jun N-terminal kinase.

PubMed

Gaillard, Pascale; Jeanclaude-Etter, Isabelle; Ardissone, Vittoria; Arkinstall, Steve; Cambet, Yves; Camps, Montserrat; Chabert, Christian; Church, Dennis; Cirillo, Rocco; Gretener, Denise; Halazy, Serge; Nichols, Anthony; Szyndralewiez, Cedric; Vitte, Pierre-Alain; Gotteland, Jean-Pierre

2005-07-14

Several lines of evidence support the hypothesis that c-Jun N-terminal kinase (JNKs) plays a critical role in a wide range of diseases including cell death (apoptosis)-related disorders (neurodegenerative diseases, brain, heart, and renal ischemia, epilepsy) and inflammatory disorders (multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases). Screening of our internal compound collection for inhibitors of JNK3 led to the identification of (benzothiazol-2-yl)acetonitrile derivatives as potent and selective JNK1, -2, -3 inhibitors. Starting from initial hit 1 (AS007149), the chemistry and initial structure-activity relationship (SAR) of this novel and unique kinase inhibitor template were explored. Investigation of the SAR rapidly revealed that the benzothiazol-2-ylacetonitrile pyrimidine core was crucial to retain a good level of potency on rat JNK3. Therefore, compound 6 was further optimized by exploring a number of distal combinations in place of the chlorine atom. This led to the observation that the presence of an aromatic group, two carbons away from the aminopyrimidine moiety and bearing substituents conferring hydrogen bond acceptor (HBA) properties, could improve the potency. Further improvements to the biological and biopharmaceutical profile of the most promising compounds were performed, resulting in the discovery of compound 59 (AS601245). The in vitro and in vivo anti-inflammatory potential of this new JNK inhibitor was investigated and found to demonstrate efficacy per oral route in an experimental model of rheumatoid arthritis (RA).

Glutathione S-Transferases Interact with AMP-Activated Protein Kinase: Evidence for S-Glutathionylation and Activation In Vitro

PubMed Central

Polge, Cécile; Ramirez, Sacnicte; Michelland, Sylvie; Sève, Michel; Vertommen, Didier; Rider, Mark; Lentze, Nicolas; Auerbach, Daniel; Schlattner, Uwe

2013-01-01

AMP-activated protein kinase (AMPK) is a cellular and whole body energy sensor with manifold functions in regulating energy homeostasis, cell morphology and proliferation in health and disease. Here we apply multiple, complementary in vitro and in vivo interaction assays to identify several isoforms of glutathione S-transferase (GST) as direct AMPK binding partners: Pi-family member rat GSTP1 and Mu-family members rat GSTM1, as well as Schistosoma japonicum GST. GST/AMPK interaction is direct and involves the N-terminal domain of the AMPK β-subunit. Complex formation of the mammalian GSTP1 and -M1 with AMPK leads to their enzymatic activation and in turn facilitates glutathionylation and activation of AMPK in vitro. GST-facilitated S-glutathionylation of AMPK may be involved in rapid, full activation of the kinase under mildly oxidative physiological conditions. PMID:23741294

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

PubMed Central

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

Effect of Tamoxifen and Brain-Penetrant Protein Kinase C and c-Jun N-Terminal Kinase Inhibitors on Tolerance to Opioid-Induced Respiratory Depression in Mice.

PubMed

Withey, Sarah L; Hill, Rob; Lyndon, Abigail; Dewey, William L; Kelly, Eamonn; Henderson, Graeme

2017-04-01

Respiratory depression is the major cause of death in opioid overdose. We have previously shown that prolonged treatment of mice with morphine induces profound tolerance to the respiratory-depressant effects of the drug (Hill et al., 2016). The aim of the present study was to investigate whether tolerance to opioid-induced respiratory depression is mediated by protein kinase C (PKC) and/or c-Jun N-terminal kinase (JNK). We found that although mice treated for up to 6 days with morphine developed tolerance, as measured by the reduced responsiveness to an acute challenge dose of morphine, administration of the brain-penetrant PKC inhibitors tamoxifen and calphostin C restored the ability of acute morphine to produce respiratory depression in morphine-treated mice. Importantly, reversal of opioid tolerance was dependent on the nature of the opioid ligand used to induce tolerance, as these PKC inhibitors did not reverse tolerance induced by prolonged treatment of mice with methadone nor did they reverse the protection to acute morphine-induced respiratory depression afforded by prolonged treatment with buprenorphine. We found no evidence for the involvement of JNK in morphine-induced tolerance to respiratory depression. These results indicate that PKC represents a major mechanism underlying morphine tolerance, that the mechanism of opioid tolerance to respiratory depression is ligand-dependent, and that coadministration of drugs with PKC-inhibitory activity and morphine (as well as heroin, largely metabolized to morphine in the body) may render individuals more susceptible to overdose death by reversing tolerance to the effects of morphine. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Effect of Tamoxifen and Brain-Penetrant Protein Kinase C and c-Jun N-Terminal Kinase Inhibitors on Tolerance to Opioid-Induced Respiratory Depression in Mice

PubMed Central

Withey, Sarah L.; Hill, Rob; Lyndon, Abigail; Dewey, William L.; Kelly, Eamonn

2017-01-01

Respiratory depression is the major cause of death in opioid overdose. We have previously shown that prolonged treatment of mice with morphine induces profound tolerance to the respiratory-depressant effects of the drug (Hill et al., 2016). The aim of the present study was to investigate whether tolerance to opioid-induced respiratory depression is mediated by protein kinase C (PKC) and/or c-Jun N-terminal kinase (JNK). We found that although mice treated for up to 6 days with morphine developed tolerance, as measured by the reduced responsiveness to an acute challenge dose of morphine, administration of the brain-penetrant PKC inhibitors tamoxifen and calphostin C restored the ability of acute morphine to produce respiratory depression in morphine-treated mice. Importantly, reversal of opioid tolerance was dependent on the nature of the opioid ligand used to induce tolerance, as these PKC inhibitors did not reverse tolerance induced by prolonged treatment of mice with methadone nor did they reverse the protection to acute morphine-induced respiratory depression afforded by prolonged treatment with buprenorphine. We found no evidence for the involvement of JNK in morphine-induced tolerance to respiratory depression. These results indicate that PKC represents a major mechanism underlying morphine tolerance, that the mechanism of opioid tolerance to respiratory depression is ligand-dependent, and that coadministration of drugs with PKC-inhibitory activity and morphine (as well as heroin, largely metabolized to morphine in the body) may render individuals more susceptible to overdose death by reversing tolerance to the effects of morphine. PMID:28130265

Mucin1 mediates autocrine transforming growth factor beta signaling through activating the c-Jun N-terminal kinase/activator protein 1 pathway in human hepatocellular carcinoma cells.

PubMed

Li, Qiongshu; Liu, Guomu; Shao, Dan; Wang, Juan; Yuan, Hongyan; Chen, Tanxiu; Zhai, Ruiping; Ni, Weihua; Tai, Guixiang

2015-02-01

In a previous study, we observed by global gene expression analysis that oncogene mucin1 (MUC1) silencing decreased transforming growth factor beta (TGF-β) signaling in the human hepatocellular carcinoma (HCC) cell line SMMC-7721. In this study, we report that MUC1 overexpression enhanced the levels of phosphorylated Smad3 linker region (p-Smad3L) (Ser-213) and its target gene MMP-9 in HCC cells, suggesting that MUC1 mediates TGF-β signaling. To investigate the effect of MUC1 on TGF-β signaling, we determined TGF-β secretion in MUC1 gene silencing and overexpressing cell lines. MUC1 expression enhanced not only TGF-β1 expression at the mRNA and protein levels but also luciferase activity driven by a TGF-β promoter, as well as elevated the activation of c-Jun N-terminal kinase (JNK) and c-Jun, a member of the activation protein 1 (AP-1) transcription factor family. Furthermore, pharmacological reduction of TGF-β receptor (TβR), JNK and c-Jun activity inhibited MUC1-induced autocrine TGF-β signaling. Moreover, a co-immunoprecipitation assay showed that MUC1 directly bound and activated JNK. In addition, both MUC1-induced TGF-β secretion and exogenous TGF-β1 significantly increased Smad signaling and cell migration, which were markedly inhibited by either TβR inhibitor or small interfering RNA silencing of TGF-β1 gene in HCC cells. The high correlation between MUC1 and TGF-β1 or p-Smad3L (Ser-213) expression was shown in tumor tissues from HCC patients by immunohistochemical staining analysis. Collectively, these results indicate that MUC1 mediates autocrine TGF-β signaling by activating the JNK/AP-1 pathway in HCC cells. Therefore, MUC1 plays a key role in HCC progression and could serve as an attractive target for HCC therapy. Copyright © 2014 Elsevier Ltd. All rights reserved.

MarvelD3 regulates the c-Jun N-terminal kinase pathway during eye development in Xenopus

PubMed Central

Vacca, Barbara; Sanchez-Heras, Elena; Steed, Emily; Balda, Maria S.; Ohnuma, Shin-Ichi; Sasai, Noriaki; Mayor, Roberto

2016-01-01

ABSTRACT Ocular morphogenesis requires several signalling pathways controlling the expression of transcription factors and cell-cycle regulators. However, despite a well-known mechanism, the dialogue between those signals and factors remains to be unveiled. Here, we identify a requirement for MarvelD3, a tight junction transmembrane protein, in eye morphogenesis in Xenopus. MarvelD3 depletion led to an abnormally pigmented eye or even an eye-less phenotype, which was rescued by ectopic MarvelD3 expression. Altering MarvelD3 expression led to deregulated expression of cell-cycle regulators and transcription factors required for eye development. The eye phenotype was rescued by increased c-Jun terminal Kinase activation. Thus, MarvelD3 links tight junctions and modulation of the JNK pathway to eye morphogenesis. PMID:27870636

The Regulatory and Kinase Domains but Not the Interdomain Linker Determine Human Double-stranded RNA-activated Kinase (PKR) Sensitivity to Inhibition by Viral Non-coding RNAs.

PubMed

Sunita, S; Schwartz, Samantha L; Conn, Graeme L

2015-11-20

Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an important component of the innate immune system that presents a crucial first line of defense against viral infection. PKR has a modular architecture comprising a regulatory N-terminal dsRNA binding domain and a C-terminal kinase domain interposed by an unstructured ∼80-residue interdomain linker (IDL). Guided by sequence alignment, we created IDL deletions in human PKR (hPKR) and regulatory/kinase domain swap human-rat chimeric PKRs to assess the contributions of each domain and the IDL to regulation of the kinase activity by RNA. Using circular dichroism spectroscopy, limited proteolysis, kinase assays, and isothermal titration calorimetry, we show that each PKR protein is properly folded with similar domain boundaries and that each exhibits comparable polyinosinic-cytidylic (poly(rI:rC)) dsRNA activation profiles and binding affinities for adenoviral virus-associated RNA I (VA RNAI) and HIV-1 trans-activation response (TAR) RNA. From these results we conclude that the IDL of PKR is not required for RNA binding or mediating changes in protein conformation or domain interactions necessary for PKR regulation by RNA. In contrast, inhibition of rat PKR by VA RNAI and TAR RNA was found to be weaker than for hPKR by 7- and >300-fold, respectively, and each human-rat chimeric domain-swapped protein showed intermediate levels of inhibition. These findings indicate that PKR sequence or structural elements in the kinase domain, present in hPKR but absent in rat PKR, are exploited by viral non-coding RNAs to accomplish efficient inhibition of PKR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

c-Jun N-terminal kinase in pancreatic tumor stroma augments tumor development in mice.

PubMed

Sato, Takeshi; Shibata, Wataru; Hikiba, Yohko; Kaneta, Yoshihiro; Suzuki, Nobumi; Ihara, Sozaburo; Ishii, Yasuaki; Sue, Soichiro; Kameta, Eri; Sugimori, Makoto; Yamada, Hiroaki; Kaneko, Hiroaki; Sasaki, Tomohiko; Ishii, Tomohiro; Tamura, Toshihide; Kondo, Masaaki; Maeda, Shin

2017-11-01

Pancreatic ductal adenocarcinoma (PDAC) is a life-threatening disease and there is an urgent need to develop improved therapeutic approaches. The role of c-Jun N-terminal kinase (JNK) in PDAC stroma is not well defined even though dense desmoplastic reactions are characteristic of PDAC histology. We aimed to explore the role of JNK in PDAC stroma in mice. We crossed Ptf1a Cre/+ ;Kras G12D/+ mice with JNK1 -/- mice to generate Ptf1a Cre/+ ;Kras G12D/+ ;JNK1 -/- (Kras;JNK1 -/- ) mice. Tumor weight was significantly lower in Kras;JNK1 -/- mice than in Kras;JNK1 +/- mice, whereas histopathological features were similar. We also transplanted a murine PDAC cell line (mPC) with intact JNK1 s.c. into WT and JNK1 -/- mice. Tumor diameters were significantly smaller in JNK1 -/- mice. Phosphorylated JNK (p-JNK) was activated in α-smooth muscle actin (SMA)-positive cells in tumor stroma, and mPC-conditioned medium activated p-JNK in tumor-associated fibroblasts (TAF) in vitro. Relative expression of Ccl20 was downregulated in stimulated TAF. Ccl20 is an important chemokine that promotes CD8 + T-cell infiltration by recruitment of dendritic cells, and the number of CD8 + T cells was decreased in Kras;JNK1 +/- mice compared with Kras;JNK1 -/- mice. These results suggest that the cancer secretome decreases Ccl20 secretion from TAF by activation of JNK, and downregulation of Ccl20 secretion might be correlated with reduction of infiltrating CD8 + T cells. Therefore, we concluded that inhibition of activated JNK in pancreatic tumor stroma could be a potential therapeutic target to increase Ccl20 secretion from TAF and induce accumulation of CD8 + T cells, which would be expected to enhance antitumor immunity. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Hydrophobic Motif Phosphorylation Coordinates Activity and Polar Localization of the Neurospora crassa Nuclear Dbf2-Related Kinase COT1

PubMed Central

Maerz, Sabine; Dettmann, Anne

2012-01-01

Nuclear Dbf2p-related (NDR) kinases and associated proteins are recognized as a conserved network that regulates eukaryotic cell polarity. NDR kinases require association with MOB adaptor proteins and phosphorylation of two conserved residues in the activation segment and hydrophobic motif for activity and function. We demonstrate that the Neurospora crassa NDR kinase COT1 forms inactive dimers via a conserved N-terminal extension, which is also required for the interaction of the kinase with MOB2 to generate heterocomplexes with basal activity. Basal kinase activity also requires autophosphorylation of the COT1-MOB2 complex in the activation segment, while hydrophobic motif phosphorylation of COT1 by the germinal center kinase POD6 fully activates COT1 through induction of a conformational change. Hydrophobic motif phosphorylation is also required for plasma membrane association of the COT1-MOB2 complex. MOB2 further restricts the membrane-associated kinase complex to the hyphal apex to promote polar cell growth. These data support an integrated mechanism of NDR kinase regulation in vivo, in which kinase activation and cellular localization of COT1 are coordinated by dual phosphorylation and interaction with MOB2. PMID:22451488

Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain

PubMed Central

Munir, Annum

2016-01-01

ABSTRACT 5′- and 3′-end-healing reactions are key steps in nucleic acid break repair in which 5′-OH ends are phosphorylated by a polynucleotide kinase (Pnk) and 3′-PO4 or 2′,3′-cyclic-PO4 ends are hydrolyzed by a phosphoesterase to generate the 5′-PO4 and 3′-OH termini required for sealing by classic polynucleotide ligases. End-healing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2′,3′-phosphoesterase HD domain and a C-terminal 5′-OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5′-OH polynucleotides (9-mers or longer) in the presence of magnesium and any nucleoside triphosphate donor. HD-Pnk dephosphorylates RNA 2′,3′-cyclic phosphate, RNA 3′-phosphate, RNA 2′-phosphate, and DNA 3′-phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper, or cobalt. HD-Pnk homologs are present in genera from 11 bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. IMPORTANCE The present study provides insights regarding the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnk as the exemplar of a novel clade of dual 5′- and 3′-end-healing enzymes that phosphorylate 5′-OH termini and dephosphorylate 2′,3′-cyclic-PO4, 3′-PO4, and 2′-PO4 ends. The distinctive feature of HD-Pnk is its domain composition, i.e., a fusion of an N-terminal HD phosphohydrolase module and a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, same domain order, and similar polypeptide sizes are distributed widely among genera from 11 bacterial phyla. PMID:27895092

The β subunit of yeast AMP-activated protein kinase directs substrate specificity in response to alkaline stress.

PubMed

Chandrashekarappa, Dakshayini G; McCartney, Rhonda R; O'Donnell, Allyson F; Schmidt, Martin C

2016-12-01

Saccharomyces cerevisiae express three isoforms of Snf1 kinase that differ by which β subunit is present, Gal83, Sip1 or Sip2. Here we investigate the abundance, activation, localization and signaling specificity of the three Snf1 isoforms. The relative abundance of these isoforms was assessed by quantitative immunoblotting using two different protein extraction methods and by fluorescence microscopy. The Gal83 containing isoform is the most abundant in all assays while the abundance of the Sip1 and Sip2 isoforms is typically underestimated especially in glass-bead extractions. Earlier studies to assess Snf1 isoform function utilized gene deletions as a means to inactivate specific isoforms. Here we use point mutations in Gal83 and Sip2 and a 17 amino acid C-terminal truncation of Sip1 to inactivate specific isoforms without affecting their abundance or association with the other subunits. The effect of low glucose and alkaline stresses was examined for two Snf1 phosphorylation substrates, the Mig1 and Mig2 proteins. Any of the three isoforms was capable of phosphorylating Mig1 in response to glucose stress. In contrast, the Gal83 isoform of Snf1 was both necessary and sufficient for the phosphorylation of the Mig2 protein in response to alkaline stress. Alkaline stress led to the activation of all three isoforms yet only the Gal83 isoform translocates to the nucleus and phosphorylates Mig2. Deletion of the SAK1 gene blocked nuclear translocation of Gal83 and signaling to Mig2. These data strongly support the idea that Snf1 signaling specificity is mediated by localization of the different Snf1 isoforms. Copyright © 2016 Elsevier Inc. All rights reserved.

Ste20-like kinase, SLK, activates the heat shock factor 1 - Hsp70 pathway.

PubMed

Cybulsky, Andrey V; Guillemette, Julie; Papillon, Joan

2016-09-01

Expression and activation of SLK increases during renal ischemia-reperfusion injury. When highly expressed, SLK signals via c-Jun N-terminal kinase and p38 to induce apoptosis, and it exacerbates apoptosis induced by ischemia-reperfusion injury. Overexpression of SLK in glomerular epithelial cells (GECs)/podocytes in vivo induces injury and proteinuria. In response to various stresses, cells enhance expression of chaperones or heat shock proteins (e.g. Hsp70), which are involved in the folding and maturation of newly synthesized proteins, and can refold denatured or misfolded proteins. We address the interaction of SLK with the heat shock factor 1 (HSF1)-Hsp70 pathway. Increased expression of SLK in GECs (following transfection) induced HSF1 transcriptional activity. Moreover, HSF1 transcriptional activity was increased by in vitro ischemia-reperfusion injury (chemical anoxia/recovery) and heat shock, and in both instances was amplified further by SLK overexpression. HSF1 binds to promoters of target genes, such as Hsp70 and induces their transcription. By analogy to HSF1, SLK stimulated Hsp70 expression. Hsp70 was also enhanced by anoxia/recovery and was further amplified by SLK overexpression. Induction of HSF1 and Hsp70 was dependent on the kinase activity of SLK, and was mediated via polo-like kinase-1. Transfection of constitutively active HSF1 enhanced Hsp70 expression and inhibited SLK-induced apoptosis. Conversely, the proapoptotic action of SLK was augmented by HSF1 shRNA, or the Hsp70 inhibitor, pifithrin-μ. In conclusion, increased expression/activity of SLK activates the HSF1-Hsp70 pathway. Hsp70 attenuates the primary proapoptotic effect of SLK. Modulation of chaperone expression may potentially be harnessed as cytoprotective therapy in renal cell injury. Copyright © 2016 Elsevier B.V. All rights reserved.

Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow.

PubMed

Berk, B C; Corson, M A; Peterson, T E; Tseng, H

1995-12-01

Fluid shear stress regulates endothelial cell function, but the signal transduction mechanisms involved in mechanotransduction remain unclear. Recent findings demonstrate that several intracellular kinases are activated by mechanical forces. In particular, members of the mitogen-activated protein (MAP) kinase family are stimulated by hyperosmolarity, stretch, and stress such as heat shock. We propose a model for mechanotransduction in endothelial cells involving calcium-dependent and calcium-independent protein kinase pathways. The calcium-dependent pathway involves activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), increases in intracellular calcium and stimulation of kinases such as calcium-calmodulin and C kinases (PKC). The calcium-independent pathway involves activation of a small GTP-binding protein and stimulation of calcium-independent PKC and MAP kinases. The calcium-dependent pathway mediates the rapid, transient response to fluid shear stress including activation of nitric oxide synthase (NOS) and ion transport. In contrast, the calcium-independent pathway mediates a slower response including the sustained activation of NOS and changes in cell morphology and gene expression. We propose that focal adhesion complexes link the calcium-dependent and calcium-independent pathways by regulating activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase (which regulates PIP2 levels) and p125 focal adhesion kinase (FAK, which phosphorylates paxillin and interacts with cytoskeletal proteins). This model predicts that dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in mechanotransduction will be integrated by calcium-dependent and calcium-independent kinases to generate intracellular signals involved in the endothelial cell response to flow.

Inhibition of protein kinase C α/βII and activation of c-Jun NH2-terminal kinase mediate glycyrrhetinic acid induced apoptosis in non-small cell lung cancer NCI-H460 cells.

PubMed

Song, Junho; Ko, Hyun-suk; Sohn, Eun Jung; Kim, Bonglee; Kim, Jung Hyo; Kim, Hee Jeong; Kim, Chulwoo; Kim, Jai-eun; Kim, Sung-Hoon

2014-02-15

Though glycyrrhetinic acid (GA) from Glycyrrhiza glabra was known to exert antioxidant, antifilarial, hepatoprotective, anti-inflammatory and anti-tumor effects, the antitumor mechanism of GA was not clearly elucidated in non-small cell lung cancer cells (NSCLCCs). Thus, in the present study, the underlying apoptotic mechanism of GA was examined in NCI-H460 NSCLCCs. GA significantly suppressed the viability of NCI-H460 and A549 non-small lung cancer cells. Also, GA significantly increased the sub G1 population by cell cycle analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells in a concentration dependent manner in NCI-H460 non-small lung cancer cells. Consistently, GA cleaved poly (ADP-ribosyl) polymerase (PARP), caspase 9/3, attenuated the expression of Bcl-XL, Bcl-2, Cyclin D1 and Cyclin E in NCI-H460 cells. Interestingly, GA attenuated the phosphorylation of protein kinase C (PKC) α/βII and extracellular activated protein kinase (ERK) as well as activated the phosphorylation of PKC δ and c-Jun NH2-terminal kinase in NCI-H460 cells. Conversely, PKC promoter phorbol 12-myristate 13-acetate (PMA) and JNK inhibitor SP600125 reversed the cleavages of caspase 3 and PARP induced by GA in NCI-H460 cells. Overall, our findings suggest that GA induces apoptosis via inhibition of PKC α/βII and activation of JNK in NCI-H460 non-small lung cancer cells as a potent anticancer candidate for lung cancer treatment. Copyright © 2013 Elsevier Ltd. All rights reserved.

A rho-binding protein kinase C-like activity is required for the function of protein kinase N in Drosophila development.

PubMed

Betson, Martha; Settleman, Jeffrey

2007-08-01

The Rho GTPases interact with multiple downstream effectors to exert their biological functions, which include important roles in tissue morphogenesis during the development of multicellular organisms. Among the Rho effectors are the protein kinase N (PKN) proteins, which are protein kinase C (PKC)-like kinases that bind activated Rho GTPases. The PKN proteins are well conserved evolutionarily, but their biological role in any organism is poorly understood. We previously determined that the single Drosophila ortholog of mammalian PKN proteins, Pkn, is a Rho/Rac-binding kinase essential for Drosophila development. By performing "rescue" studies with various Pkn mutant constructs, we have defined the domains of Pkn required for its role during Drosophila development. These studies suggested that Rho, but not Rac binding is important for Pkn function in development. In addition, we determined that the kinase domain of PKC53E, a PKC family kinase, can functionally substitute for the kinase domain of Pkn during development, thereby exemplifying the evolutionary strategy of "combining" functional domains to produce proteins with distinct biological activities. Interestingly, we also identified a requirement for Pkn in wing morphogenesis, thereby revealing the first postembryonic function for Pkn.

Upregulation of RhoB via c-Jun N-terminal kinase signaling induces apoptosis of the human gastric carcinoma NUGC-3 cells treated with NSC12618.

PubMed

Kim, Bo-Kyung; Kim, Hwan Mook; Chung, Kyung-Sook; Kim, Dong-Myung; Park, Song-Kyu; Song, Alexander; Won, Kyoung-Jae; Lee, Kiho; Oh, Yu-Kyoung; Lee, Kyeong; Song, Kyung-Bin; Simon, Julian A; Han, Gyoonhee; Won, Misun

2011-03-01

RhoB expression is reduced in most invasive tumors, with loss of RhoB expression correlating significantly with tumor stage. Here, we demonstrate that upregulation of RhoB by the potent anticancer agent NSC126188 induces apoptosis of NUGC-3 human gastric carcinoma cells. The crucial role of RhoB in NSC126188-induced apoptosis is indicated by the rescue of NUGC-3 cells from apoptosis by knockdown of RhoB. In the presence of NSC126188, c-Jun N-terminal kinase (JNK) signaling was activated, and the JNK inhibitor SP600125 reduced RhoB expression and suppressed the apoptosis of NUGC-3 cells. Knockdowns of mitogen-activated protein kinase kinase (MKK) 4/7, JNK1/2 and c-Jun downregulated RhoB expression and rescued cells from apoptotic death in the presence of NSC126188. The JNK inhibitor SP600125 suppressed transcriptional activation of RhoB in the presence of NSC126188, as indicated by a reporter assay that used luciferase under the RhoB promoter. The ability of NSC126188 to increase luciferase activity through both the p300-binding site and the inverted CCAAT sequence (iCCAAT box) suggests that JNK signaling to upregulate RhoB expression is mediated through both the p300-binding site and the iCCAAT box. However, the JNK inhibitor SP600125 did not inhibit the upregulation of RhoB by farnesyltransferase inhibitor (FTI)-277. The p300-binding site did not affect activation of the RhoB promoter by FTI-277 in NUGC-3 cells, suggesting that the transcriptional activation of RhoB by NSC126188 occurs by a different mechanism than that reported for FTIs. Our data indicate that NSC126188 increases RhoB expression via JNK-mediated signaling through a p300-binding site and iCCAAT box resulting in apoptosis of NUGC-3 cells.

c-Jun N-terminal kinase inhibitors: a patent review (2010 - 2014).

PubMed

Gehringer, Matthias; Muth, Felix; Koch, Pierre; Laufer, Stefan A

2015-01-01

c-Jun N-terminal kinases (JNKs) are involved in the emergence and progression of diverse pathologies such as neurodegenerative, cardiovascular and metabolic disorders as well as inflammation and cancer. In recent years, several highly selective pan-JNK inhibitors have been characterized and three chemical entities targeting JNKs have been investigated in clinical trials. This review summarizes patents claiming inhibitors of all JNK isoforms published between 2010 and 2014. Although primarily focusing on the patent literature, relevant peer-reviewed publications related to the covered patents have also been included. Moreover, key patents claiming novel applications of previously published chemical entities are reviewed. The article highlights a total of 28 patents from nine pharmaceutical companies and academic research groups. Although some selective pan-JNK inhibitors with reasonable in vivo profiles are now available, little is known about the isoform selectivity required for each particular indication and the development of isoform-selective JNK inhibitors still represents a challenge in JNK drug discovery. Moreover, isoform-selective tool compounds are a prerequisite to a comprehensive understanding of the biology of each JNK isoform. Potential approaches towards such compounds include the design of type-II and type-I(1)/2 binders, which are absent in the current JNK inhibitor portfolios, as well as the design of novel allosteric inhibitors. Furthermore, covalent inhibition, which already led to the first high-quality probe for JNKs, might be further exploited for gaining selectivity and in vivo efficacy. With regard to a potential therapeutic application, the recently proposed concept of covalent reversible inhibitors is expected to be attractive.

Cigarette Smoke-induced Left Ventricular Remodelling is Associated with Activation of Mitogen-activated Protein Kinases

PubMed Central

Gu, Lianzhi; Pandey, Vikas; Geenen, David L.; Chowdhury, Shamim A. K.; Piano, Mariann R.

2008-01-01

Aim To determine the effects of cigarette smoke (CS) exposure on the expression/activation of mitogen-activated protein kinases (MAPKs) (extracellular signal-regulated kinase [ERK1/2], p38-kinase [p38] and c-Jun NH2–terminal protein kinase [JNK]), norepinephrine (NE) levels and myocardial structure and function. Methods Rats were randomised to two groups: CS–exposed (n = 10) or room air (CON) (n = 12). After 5 weeks, the animals underwent echocardiography with pulse-wave Doppler flow measurements. Hearts were removed for microscopy and Western blot analysis. Results CS exposure was associated with significant increases in NE urinary levels and larger ventricular dimensions (mm) (CON = left ventricular end diastolic dimension [LVEDD] 7.99 ± 0.10, LV end systolic dimension [LVESD] 4.55 ± 0.20, CS = LVEDD 8.3 ± 0.10, LVESD 5.3 ± 0.09, p = 0.026, p = 0.003). There was also evidence of systolic dysfunction in the CS-exposed group compared to the CON group (fractional shortening %, CON = 43 ± 2, CS = 36 ± .09, p = 0.010). In CS-exposed hearts, significant increases in phosphorylated p38/total p38 (0.975 ± 0.05) and phosphorylated ERK1/2/totalERK1/2 (1.919 ± 0.050) were found compared to CON hearts (0.464 ± 0.008, 0.459 ± 0.050, respectively). No significant differences were found in JNK levels between the groups. Conclusions Increased NE levels and MAPK activation are associated with CS-related left ventricular remodelling. PMID:18815071

N-terminal regions of Mps1 kinase determine functional bifurcation.

PubMed

Araki, Yasuhiro; Gombos, Linda; Migueleti, Suellen P S; Sivashanmugam, Lavanya; Antony, Claude; Schiebel, Elmar

2010-04-05

Mps1 is a conserved kinase that in budding yeast functions in duplication of the spindle pole body (SPB), spindle checkpoint activation, and kinetochore biorientation. The identity of Mps1 targets and the subdomains that convey specificity remain largely unexplored. Using a novel combination of systematic deletion analysis and chemical biology, we identified two regions within the N terminus of Mps1 that are essential for either SPB duplication or kinetochore biorientation. Suppression analysis of the MPS1 mutants defective in SPB duplication and biochemical enrichment of Mps1 identified the essential SPB components Spc29 and the yeast centrin Cdc31 as Mps1 targets in SPB duplication. Our data suggest that phosphorylation of Spc29 by Mps1 in G1/S recruits the Mps2-Bbp1 complex to the newly formed SPB to facilitate its insertion into the nuclear envelope. Mps1 phosphorylation of Cdc31 at the conserved T110 residue controls substrate binding to Kar1 protein. These findings explain the multiple SPB duplication defects of mps1 mutants on a molecular level.

Explorations of Substituted Urea Functionality for Discovery of New Activators of the Heme Regulated Inhibitor Kinase

PubMed Central

Chen, Ting; Takrouri, Khuloud; Hee-Hwang, Sung; Rana, Sandeep; Yefidoff-Freedman, Revital; Halperin, Jose; Natarajan, Amarnath; Morisseau, Christophe; Hammock, Bruce; Chorev, Michael; Aktas, Bertal H.

2014-01-01

Heme-regulated inhibitor kinase (HRI), an eukaryotic translation initiation factor 2 alpha (eIF2α) kinase, plays critical roles in cell proliferation, differentiation, and adaptation to cytoplasmic stress. HRI is also a critical modifier of hemoglobin disorders such as β-thalassemia. We previously identified N,N′-diarylureas as potent activators of HRI suitable for studying biology of this important kinase. To expand the repertoire of chemotypes that activate HRI we screened a ~1,900 member N,N′-disubstituted urea library in the surrogate eIF2α phosphorylation assay identifying N-aryl,N′-cyclohexylphenoxyurea as a promising scaffold. We validated hit compounds as a bona-fide HRI activators in secondary assays and explored contributions of substitutions on the N-aryl and N′-cyclohexylphenoxy groups to their activity by studying focused libraries of complementing analogs. We tested these N-aryl,N′-cyclohexylphenoxyureas in the surrogate eIF2α phosphorylation and cell proliferation assays, demonstrating significantly improved bioactivities and specificities. We consider these compounds to represent lead candidates for the development of potent and specific HRI activators. PMID:24261904

4-Phenylbutyric acid reduces endoplasmic reticulum stress, trypsin activation, and acinar cell apoptosis while increasing secretion in rat pancreatic acini.

PubMed

Malo, Antje; Krüger, Burkhard; Göke, Burkhard; Kubisch, Constanze H

2013-01-01

Endoplasmic reticulum (ER) stress leads to misfolded proteins inside the ER and initiates unfolded protein response (UPR). Unfolded protein response components are involved in pancreatic function and activated during pancreatitis. However, the exact role of ER stress in the exocrine pancreas is unclear. The present study examined the effects of 4-phenylbutyric acid (4-PBA), an ER chaperone, on acini and UPR components. Rat acini were stimulated with cholecystokinin (10 pmol/L to 10 nmol/L) with or without preincubation of 4-PBA. The UPR components were analyzed, including chaperone-binding protein, protein kinaselike ER kinase, X-box-binding protein 1, c-Jun NH(2)-terminal kinase, CCAAT/enhancer-binding protein homologous protein, caspase 3, and apoptosis. Effects of 4-PBA were measured on secretion, calcium, and trypsin activation. 4-Phenylbutyric acid led to an increase of secretion, whereas trypsin activation with supraphysiological cholecystokinin was significantly reduced. 4-Phenylbutyric acid prevented chaperone-binding protein up-regulation, diminished protein kinaselike ER kinase, and c-Jun NH2-terminal kinase phosphorylation, prohibited X-box-binding protein 1 splicing and CCAAT/enhancer-binding protein homologous protein expression, caspase 3 activation, and apoptosis caused by supraphysiological cholecystokinin. By incubation with 4-PBA, beneficial in urea cycle deficiency, it was possible to enhance enzyme secretion to suppress trypsin activation, UPR activation, and proapoptotic pathways. The data hint new perspectives for the use of chemical chaperones in pancreatic diseases.

Eckol protects V79-4 lung fibroblast cells against gamma-ray radiation-induced apoptosis via the scavenging of reactive oxygen species and inhibiting of the c-Jun NH(2)-terminal kinase pathway.

PubMed

Zhang, Rui; Kang, Kyoung Ah; Piao, Mei Jing; Ko, Dong Ok; Wang, Zhi Hong; Lee, In Kyung; Kim, Bum Joon; Jeong, Il Yun; Shin, Taekyun; Park, Jae Woo; Lee, Nam Ho; Hyun, Jin Won

2008-09-04

The radioprotective effect of eckol against gamma-ray radiation-induced oxidative stress and its possible protective mechanisms were investigated. Eckol was found to reduce the intracellular reactive oxygen species generated by gamma-ray radiation. Moreover, eckol also protected against radiation-induced cellular DNA damage and membrane lipid peroxidation, which are the main targets of radiation-induced damage. In addition, eckol recovered the cell viability damaged by radiation via the inhibition of apoptosis. Irradiated cells with eckol treatment reduced the expression of bax, the activation of caspase 9 and caspase 3, which were induced by radiation. However, irradiated cells with eckol recovered the expression of bcl-2 and mitochondrial cytochrome c which were decreased by radiation. The anti-apoptotic effect of eckol exerted via the inhibition of mitogen-activated protein kinase kinase-4 (MKK4/SEK1)-c-Jun NH(2)-terminal kinase (JNK)-activator protein 1 (AP-1) cascades induced by radiation. In summary, the results suggest that eckol protects cells against the oxidative stress induced by radiation via the reduction of reactive oxygen species and the attenuation of activation in SEK1-JNK-AP-1 pathway.

Coiled-Coil Antagonism Regulates Activity of Venus Flytrap-Domain-Containing Sensor Kinases of the BvgS Family

PubMed Central

Lesne, Elodie; Dupré, Elian; Lensink, Marc F.; Locht, Camille

2018-01-01

ABSTRACT Bordetella pertussis controls the expression of its virulence regulon through the two-component system BvgAS. BvgS is a prototype for a family of multidomain sensor kinases. In BvgS, helical linkers connect periplasmic Venus flytrap (VFT) perception domains to a cytoplasmic Per-Arnt-Sim (PAS) domain and the PAS domain to the dimerization/histidine phosphotransfer (DHp) domain of the kinase. The two linkers can adopt coiled-coil structures but cannot do so simultaneously. The first linker forms a coiled coil in the kinase mode and the second in the phosphatase mode, with the other linker in both cases showing an increase in dynamic behavior. The intervening PAS domain changes its quaternary structure between the two modes. In BvgS homologues without a PAS domain, a helical “X” linker directly connects the VFT and DHp domains. Here, we used BvgS as a platform to characterize regulation in members of the PAS-less subfamily. BvgS chimeras of homologues with natural X linkers displayed various regulation phenotypes. We identified two distinct coiled-coil registers in the N- and C-terminal portions of the X linkers. Stable coil formation in the C-terminal moiety determines the phosphatase mode, similarly to BvgS; in contrast, coil formation in the N-terminal moiety along the other register leads to the kinase mode. Thus, antagonism between two registers in the VFT-DHp linker forms the basis for activity regulation in the absence of the PAS domain. The N and C moieties of the X linker play roles similar to those played by the two independent linkers in sensor kinases with a PAS domain, providing a unified mechanism of regulation for the entire family. PMID:29487240

Structure-function similarities between a plant receptor-like kinase and the human interleukin-1 receptor-associated kinase-4.

PubMed

Klaus-Heisen, Dörte; Nurisso, Alessandra; Pietraszewska-Bogiel, Anna; Mbengue, Malick; Camut, Sylvie; Timmers, Ton; Pichereaux, Carole; Rossignol, Michel; Gadella, Theodorus W J; Imberty, Anne; Lefebvre, Benoit; Cullimore, Julie V

2011-04-01

Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology modeling revealed that the plant RLK contains structural features particular to this group of kinases, including the tyrosine gatekeeper and the N-terminal extension α-helix B. Functional analysis revealed the importance of these conserved features for kinase activity and suggests that kinase activity is essential for the biological role of LYK3 in the establishment of the root nodule nitrogen-fixing symbiosis with rhizobia bacteria. The kinase domain of LYK3 has dual serine/threonine and tyrosine specificity, and mass spectrometry analysis identified seven serine, eight threonine, and one tyrosine residue as autophosphorylation sites in vitro. Three activation loop serine/threonine residues are required for biological activity, and molecular dynamics simulations suggest that Thr-475 is the prototypical phosphorylated residue that interacts with the conserved arginine in the catalytic loop, whereas Ser-471 and Thr-472 may be secondary sites. A threonine in the juxtamembrane region and two threonines in the C-terminal lobe of the kinase domain are important for biological but not kinase activity. We present evidence that the structure-function similarities that we have identified between LYK3 and IRAK-4 may be more widely applicable to plant RLKs in general.

Novel Autophosphorylation Sites of Src Family Kinases Regulate Kinase Activity and SH2 Domain Binding Capacity

PubMed Central

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

2016-01-01

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

Structure and substrate recruitment of the human spindle checkpoint kinase Bub1.

PubMed

Kang, Jungseog; Yang, Maojun; Li, Bing; Qi, Wei; Zhang, Chao; Shokat, Kevan M; Tomchick, Diana R; Machius, Mischa; Yu, Hongtao

2008-11-07

In mitosis, the spindle checkpoint detects a single unattached kinetochore, inhibits the anaphase-promoting complex or cyclosome (APC/C), and prevents premature sister chromatid separation. The checkpoint kinase Bub1 contributes to checkpoint sensitivity through phosphorylating the APC/C activator, Cdc20, and inhibiting APC/C catalytically. We report here the crystal structure of the kinase domain of Bub1, revealing the requirement of an N-terminal extension for its kinase activity. Though the activation segment of Bub1 is ordered and has structural features indicative of active kinases, the C-terminal portion of this segment sterically restricts substrate access to the active site. Bub1 uses docking motifs, so-called KEN boxes, outside its kinase domain to recruit Cdc20, one of two known KEN box receptors. The KEN boxes of Bub1 are required for the spindle checkpoint in human cells. Therefore, its unusual active-site conformation and mode of substrate recruitment suggest that Bub1 has an exquisitely tuned specificity for Cdc20.

BDE-47 induces oxidative stress, activates MAPK signaling pathway, and elevates de novo lipogenesis in the copepod Paracyclopina nana.

PubMed

Lee, Min-Chul; Puthumana, Jayesh; Lee, Seung-Hwi; Kang, Hye-Min; Park, Jun Chul; Jeong, Chang-Bum; Han, Jeonghoon; Hwang, Dae-Sik; Seo, Jung Soo; Park, Heum Gi; Om, Ae-Son; Lee, Jae-Seong

2016-12-01

Brominated flame retardant, 2, 2', 4, 4'-tetrabromodiphenyl ether (BDE-47), has received grave concerns as a persistent organic pollutant, which is toxic to marine organisms, and a suspected link to endocrine abnormalities. Despite the wide distribution in the marine ecosystem, very little is known about the toxic impairments on marine organisms, particularly on invertebrates. Thus, we examined the adverse effects of BDE-47 on life history trait (development), oxidative markers, fatty acid composition, and lipid accumulation in response to BDE-47-induced stress in the marine copepod Paracyclopina nana. Also, activation level of mitogen-activated protein kinase (MAPK) signaling pathways along with the gene expression profile of de novo lipogenesis (DNL) pathways were addressed. As a result, BDE-47 induced oxidative stress (e.g. reactive oxygen species, ROS) mediated activation of extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK) signaling cascades in MAPK pathways. Activated MAPK pathways, in turn, induced signal molecules that bind to the transcription factors (TFs) responsible for lipogenesis to EcR, SREBP, ChREBP promoters. Also, the stress stimulated the conversion of saturated fatty acids (SFAs) to polyunsaturated fatty acids (PUFAs), a preparedness of the organism to adapt the observed stress, which could be correlated with the elongase and desaturase gene (e.g. ELO3, Δ5-DES, Δ9-DES) expressions, and then extended to the delayed early post-embryonic development and increased accumulation of lipid droplets in P. nana. This study will provide a better understanding of how BDE-47 effects on marine invertebrates particularly on the copepods, an important link in the marine food chain. Copyright © 2016 Elsevier B.V. All rights reserved.

Stimulation of spinal dorsal horn β2-adrenergic receptor ameliorates neuropathic mechanical hypersensitivity through a reduction of phosphorylation of microglial p38 MAP kinase and astrocytic c-jun N-terminal kinase.

PubMed

Zhang, Fang Fang; Morioka, Norimitsu; Abe, Hiromi; Fujii, Shiori; Miyauchi, Kazuki; Nakamura, Yoki; Hisaoka-Nakashima, Kazue; Nakata, Yoshihiro

2016-12-01

The noradrenaline-adrenergic system has a crucial role in controlling nociceptive transduction at the spinal level. While α-adrenergic receptors are known to regulate nociceptive neurotransmitter release at the spinal presynaptic level, it is not entirely clear whether β-adrenergic receptors are involved in controlling pain transduction at the spinal level as well. The current study elucidated a role of β-adrenergic receptors in neuropathic pain in mice following a partial sciatic nerve ligation (PSNL). In addition, the cellular and intracellular signaling cascade induced by β-adrenergic receptors in neuropathic mice was elaborated. Intrathecal injection of isoproterenol (1 nmol), a nonselective β-adrenergic receptor agonist, briefly ameliorated hind paw mechanical hypersensitivity of PSNL mice. Isoproterenol's antinociceptive effect was mediated through β2-adrenergic receptors since pretreatment with ICI118551, a selective β2-adrenergic receptor antagonist, but not with CGP20712A, a selective β1-adrenergic receptor antagonist, significantly attenuated isoproterenol's effect. Furthermore, intrathecal treatment with a selective β2-adrenergic receptor agonist, terbutaline, but not a selective β1-adrenergic receptor agonist, dobutamine, also significantly ameliorated neuropathic pain. Fourteen days after PSNL, increased phosphorylation of both p38 Mitogen-activated protein kinase (MAPK) in microglia and c-jun N-terminal kinase (JNK) in astrocytes of ipsilateral spinal dorsal horn were observed. Phosphorylation of both microglial p38 MAPK and astrocytic JNK were downregulated by stimulation of the β2-adrenergic receptor. Together, these results suggest that spinal β2-adrenergic receptor have an inhibitory role in neuropathic nociceptive transduction at the spinal level through a downregulation of glial activity, perhaps through modulation of MAP kinases phosphorylation. Thus, targeting of β2-adrenergic receptors could be an effective therapeutic strategy

Coenzyme Q10 inhibits the release of glutamate in rat cerebrocortical nerve terminals by suppression of voltage-dependent calcium influx and mitogen-activated protein kinase signaling pathway.

PubMed

Chang, Yi; Huang, Shu-Kuei; Wang, Su-Jane

2012-12-05

This study investigates the effects and possible mechanism of coenzyme Q10 (CoQ10) on endogenous glutamate release in the cerebral cortex nerve terminals of rats. CoQ10 inhibited the release of glutamate evoked by the K+ channel blocker 4-aminopyridine (4-AP). CoQ10 reduced the depolarization-induced increase in cytosolic [Ca2+]c but did not alter the 4-AP-mediated depolarization. The effect of CoQ10 on evoked glutamate release was abolished by blocking the Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2+ channels and mitogen-activated protein kinase kinase (MEK). In addition, CoQ10 decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synaptic vesicle-associated protein synapsin I, a major presynaptic substrate for ERK. Moreover, the inhibition of glutamate release by CoQ10 was strongly attenuated in mice without synapsin I. These results suggest that CoQ10 inhibits glutamate release from cortical synaptosomes in rats through the suppression of the presynaptic voltage-dependent Ca2+ entry and ERK/synapsin I signaling pathway.

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

PubMed Central

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

2007-01-01

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

Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis

NASA Technical Reports Server (NTRS)

Huang, Yafan; Li, Hui; Hutchison, Claire E.; Laskey, James; Kieber, Joseph J.

2003-01-01

CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1-8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1-8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.

Ca2+-independent Activation of Ca2+/Calmodulin-dependent Protein Kinase II Bound to the C-terminal Domain of CaV2.1 Calcium Channels*

PubMed Central

Magupalli, Venkat G.; Mochida, Sumiko; Yan, Jin; Jiang, Xin; Westenbroek, Ruth E.; Nairn, Angus C.; Scheuer, Todd; Catterall, William A.

2013-01-01

Ca2+/calmodulin-dependent protein kinase II (CaMKII) forms a major component of the postsynaptic density where its functions in synaptic plasticity are well established, but its presynaptic actions are poorly defined. Here we show that CaMKII binds directly to the C-terminal domain of CaV2.1 channels. Binding is enhanced by autophosphorylation, and the kinase-channel signaling complex persists after dephosphorylation and removal of the Ca2+/CaM stimulus. Autophosphorylated CaMKII can bind the CaV2.1 channel and synapsin-1 simultaneously. CaMKII binding to CaV2.1 channels induces Ca2+-independent activity of the kinase, which phosphorylates the enzyme itself as well as the neuronal substrate synapsin-1. Facilitation and inactivation of CaV2.1 channels by binding of Ca2+/CaM mediates short term synaptic plasticity in transfected superior cervical ganglion neurons, and these regulatory effects are prevented by a competing peptide and the endogenous brain inhibitor CaMKIIN, which blocks binding of CaMKII to CaV2.1 channels. These results define the functional properties of a signaling complex of CaMKII and CaV2.1 channels in which both binding partners are persistently activated by their association, and they further suggest that this complex is important in presynaptic terminals in regulating protein phosphorylation and short term synaptic plasticity. PMID:23255606

Abl N-terminal Cap stabilization of SH3 domain dynamics†

PubMed Central

Chen, Shugui; Dumitrescu, Teodora Pene; Smithgall, Thomas E.; Engen, John R.

2008-01-01

Crystal structures and other biochemical data indicate that the N-terminal cap (NCap) region of the Abelson tyrosine kinase (c-Abl) is important for maintaining the downregulated conformation of the kinase domain. The exact contributions that NCap makes in stabilizing the various intramolecular interactions within c-Abl are less clear. While the NCap appears important for locking the SH3/SH2 domains to the back of the kinase domain, there may be other more subtle elements of regulation. Hydrogen exchange (HX) and mass spectrometry (MS) were used to determine if the NCap contributes to intramolecular interactions involving the Abl SH3 domain. Under physiological conditions, the Abl SH3 domain underwent partial unfolding and its unfolding half-life was slowed during binding to the SH2-kinase linker, providing a unique assay to test NCap-induced stabilization of the SH3 domain in various constructs. The results showed that NCap stabilizes the dynamics of the SH3 domain in certain constructs but does not increase the relative affinity of the SH3 domain for the native SH2-kinase linker. The stabilization effect was absent in constructs of just NCap + SH3 but was obvious when the SH2 domain and the SH2-kinase linker were present. These results suggest that interactions between NCap and the SH3 domain can contribute to c-Abl stabilization in constructs that contain at least the SH2 domain, an effect that may partially compensate for the absence of the negative regulatory C-terminal tail found in the related Src family of kinases. PMID:18452309

Mitogen- and Stress-Activated Protein Kinase 1 Regulates Status Epilepticus-Evoked Cell Death in the Hippocampus

PubMed Central

Choi, Yun-Sik; Horning, Paul; Aten, Sydney; Karelina, Kate; Alzate-Correa, Diego; Arthur, J. Simon C.; Hoyt, Kari R.; Obrietan, Karl

2017-01-01

Mitogen-activated protein kinase (MAPK) signaling has been implicated in a wide range of neuronal processes, including development, plasticity, and viability. One of the principal downstream targets of both the extracellular signal-regulated kinase/MAPK pathway and the p38 MAPK pathway is Mitogen- and Stress-activated protein Kinase 1 (MSK1). Here, we sought to understand the role that MSK1 plays in neuroprotection against excitotoxic stimulation in the hippocampus. To this end, we utilized immunohistochemical labeling, a MSK1 null mouse line, cell viability assays, and array-based profiling approaches. Initially, we show that MSK1 is broadly expressed within the major neuronal cell layers of the hippocampus and that status epilepticus drives acute induction of MSK1 activation. In response to the status epilepticus paradigm, MSK1 KO mice exhibited a striking increase in vulnerability to pilocarpine-evoked cell death within the CA1 and CA3 cell layers. Further, cultured MSK1 null neurons exhibited a heighted level of N-methyl-D-aspartate-evoked excitotoxicity relative to wild-type neurons, as assessed using the lactate dehydrogenase assay. Given these findings, we examined the hippocampal transcriptional profile of MSK1 null mice. Affymetrix array profiling revealed that MSK1 deletion led to the significant (>1.25-fold) downregulation of 130 genes and an upregulation of 145 genes. Notably, functional analysis indicated that a subset of these genes contribute to neuroprotective signaling networks. Together, these data provide important new insights into the mechanism by which the MAPK/MSK1 signaling cassette confers neuroprotection against excitotoxic insults. Approaches designed to upregulate or mimic the functional effects of MSK1 may prove beneficial against an array of degenerative processes resulting from excitotoxic insults. PMID:28870089

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

PubMed

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

1997-09-01

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

CD40 inhibits replication of hepatitis C virus in primary human hepatocytes by c-Jun N terminal kinase activation independent from the interferon pathway.

PubMed

Rau, Sibylle J; Hildt, Eberhard; Himmelsbach, Kiyoshi; Thimme, Robert; Wakita, Takaji; Blum, Hubert E; Fischer, Richard

2013-01-01

CD40, a member of the tumor necrosis factor receptor family, and its ligand, CD40L (CD154), are important regulators of the antiviral immune response. CD40L is up-regulated on lymphocytes and CD40 on hepatocytes during infection with hepatitis C virus (HCV); we investigated the role of CD40 signaling during HCV replication in hepatocytes. Viral replication was studied in primary human hepatocytes (PHH) and Huh7.5 cells using the infectious HCV Japanese fulminate hepatitis 1 isolate (JFH1) culture system, and in coculture with HCV antigen-specific CD8+ T cells. CD40L rapidly and transiently inhibits expression of the HCV nonstructural proteins NS3 and NS5A as well as HCV structural proteins core and E2 in Huh7.5 cells. Similarly, CD40L prevented replication of HCV in PHH, in synergy with interferon (IFN)-alpha. In Huh7.5 cells with replicating HCV, CD40L prevented production of infectious viral particles. When HCV antigen-specific CD8+ T cells were cocultured with HLA-A2-expressing Huh7 cells that had replicating virus, the T cells became activated, up-regulated CD40L, and inhibited HCV replication. Inhibition of CD40L partially prevented the antiviral activity of the CD8+ T cells. The antiviral effect of CD40L required activation of c-Jun N terminal kinases (JNK)1/2, but not induction of apoptosis or the JAK/STAT pathway that is necessary for the antiviral effects of IFNs. CD40 inhibits HCV replication by a novel, innate immune mechanism. This pathway might mediate viral clearance, and disruptions might be involved in the pathogenesis of HCV infection. Copyright © 2012 American Association for the Study of Liver Diseases.

Evolutionarily conserved IMPACT impairs various stress responses that require GCN1 for activating the eIF2 kinase GCN2

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

Cambiaghi, Tavane D.; Pereira, Catia M.; Shanmugam, Renuka

2014-01-10

Highlights: •GCN1 is required for mammalian and yeast GCN2 function in a variety of conditions. •Mammalian IMPACT competes with GCN2 for GCN1 binding. •IMPACT and its yeast counterpart YIH1 downregulate GCN1-dependent GCN2 activation. -- Abstract: In response to a range of environmental stresses, phosphorylation of the alpha subunit of the translation initiation factor 2 (eIF2α) represses general protein synthesis coincident with increased translation of specific mRNAs, such as those encoding the transcription activators GCN4 and ATF4. The eIF2α kinase GCN2 is activated by amino acid starvation by a mechanism involving GCN2 binding to an activator protein GCN1, along with associationmore » with uncharged tRNA that accumulates during nutrient deprivation. We previously showed that mammalian IMPACT and its yeast ortholog YIH1 bind to GCN1, thereby preventing GCN1 association with GCN2 and stimulation of this eIF2α kinase during amino acid depletion. GCN2 activity is also enhanced by other stresses, including proteasome inhibition, UV irradiation and lack of glucose. Here, we provide evidence that IMPACT affects directly and specifically the activation of GCN2 under these stress conditions in mammalian cells. We show that activation of mammalian GCN2 requires its interaction with GCN1 and that IMPACT promotes the dissolution of the GCN2–GCN1 complex. To a similar extent as the overexpression of YIH1, overexpression of IMPACT in yeast cells inhibited growth under all stress conditions that require GCN2 and GCN1 for cell survival, including exposure to acetic acid, high levels of NaCl, H{sub 2}O{sub 2} or benomyl. This study extends our understanding of the roles played by GCN1 in GCN2 activation induced by a variety of stress arrangements and suggests that IMPACT and YIH1 use similar mechanisms for regulating this eIF2α kinase.« less

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

PubMed

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

2016-04-01

Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. © 2016 Federation of European Biochemical Societies.

Systemic N-terminal fragments of adrenocorticotropin reduce inflammation- and stress-induced anhedonia in rats.

PubMed

Markov, Dmitrii D; Yatsenko, Ksenia A; Inozemtseva, Lyudmila S; Grivennikov, Igor A; Myasoedov, Nikolai F; Dolotov, Oleg V

2017-08-01

Emerging evidence implicates impaired self-regulation of the hypothalamic-pituitary-adrenal (HPA) axis and inflammation as important and closely related components of the pathophysiology of major depression. Antidepressants show anti-inflammatory effects and are suggested to enhance glucocorticoid feedback inhibition of the HPA axis. HPA axis activity is also negatively self-regulated by the adrenocorticotropic hormone (ACTH), a potent anti-inflammatory peptide activating five subtypes of melanocortin receptors (MCRs). There are indications that ACTH-mediated feedback can be activated by noncorticotropic N-terminal ACTH fragments such as a potent anti-inflammatory MC1/3/4/5R agonist α-melanocyte-stimulating hormone (α-MSH), corresponding to ACTH(1-13), and a MC3/5R agonist ACTH(4-10). We investigated whether intraperitoneal administration of rats with these peptides affects anhedonia, which is a core symptom of depression. Inflammation-related anhedonia was induced by a single intraperitoneal administration of a low dose (0.025mg/kg) of lipopolysaccharide (LPS). Stress-related anhedonia was induced by the chronic unpredictable stress (CUS) procedure. The sucrose preference test was used to detect anhedonia. We found that ACTH(4-10) pretreatment decreased LPS-induced increase in serum corticosterone and tumor necrosis factor (TNF)-α, and a MC3/4R antagonist SHU9119 blocked this effect. Both α-MSH and ACTH(4-10) alleviated LPS-induced anhedonia. In the CUS model, these peptides reduced anhedonia and normalized body weight gain. The data indicate that systemic α-MSH and ACTH(4-10) produce an antidepressant-like effect on anhedonia induced by stress or inflammation, the stimuli that trigger the release of ACTH and α-MSH into the bloodstream. The results suggest a counterbalancing role of circulating melanocortins in depression and point to a new approach for antidepressant treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biophysical characterization of the calmodulin-like domain of Plasmodium falciparum calcium dependent protein kinase 3

PubMed Central

Andresen, Cecilia; Niklasson, Markus; Cassman Eklöf, Sofie; Wallner, Björn

2017-01-01

Calcium dependent protein kinases are unique to plants and certain parasites and comprise an N-terminal segment and a kinase domain that is regulated by a C-terminal calcium binding domain. Since the proteins are not found in man they are potential drug targets. We have characterized the calcium binding lobes of the regulatory domain of calcium dependent protein kinase 3 from the malaria parasite Plasmodium falciparum. Despite being structurally similar, the two lobes differ in several other regards. While the monomeric N-terminal lobe changes its structure in response to calcium binding and shows global dynamics on the sub-millisecond time-scale both in its apo and calcium bound states, the C-terminal lobe could not be prepared calcium-free and forms dimers in solution. If our results can be generalized to the full-length protein, they suggest that the C-terminal lobe is calcium bound even at basal levels and that activation is caused by the structural reorganization associated with binding of a single calcium ion to the N-terminal lobe. PMID:28746405

Seasonal variations of cellular stress response of the gilthead sea bream (Sparus aurata).

PubMed

Feidantsis, Konstantinos; Antonopoulou, Efthimia; Lazou, Antigone; Pörtner, Hans O; Michaelidis, Basile

2013-07-01

The present study aimed to investigate the seasonal cellular stress response in vital organs, like the heart, the liver, the whole blood and the skeletal (red and white) muscles of the Mediterranean fish Sparus aurata during a 1-year acclimatization period in the field, in two examined depths (0-2 m and 10-12 m). Processes studied included heat shock protein expression and protein kinase activation. Molecular responses were addressed through the expression of Hsp70 and Hsp90, the phosphorylation of stress-activated protein kinases and particularly p38 mitogen-activated protein kinase (p38 MAPK), the extracellular signal-regulated kinases (ERK-1/2) and c-Jun N-terminal kinases (JNK1/2/3). The induction of Hsp70 and Hsp90 and the phosphorylation of p38 MAPK, JNKs and ERKs in the examined five tissues of the gilthead sea bream indicated a cellular stress response under the prism of a seasonal pattern which was characterized by distinct tissue specificity. Specifically, Hsp induction and MAPK activation occurred before peak summer water temperatures, with no further increases in their levels despite increases in water temperatures. Moreover, although water temperature did not vary significantly with depth of immersion, significant effects of depth on cellular stress response were observed, probably caused by different light regime. The expression and the activation of these certain proteins can be used as tools to define the extreme thermal limits of the gilthead sea bream.

Activation of PI3K/Akt signaling by n-terminal SH2 domain mutants of the p85α regulatory subunit of PI3K is enhanced by deletion of its c-terminal SH2 domain.

PubMed

Hofmann, Bianca T; Jücker, Manfred

2012-10-01

The phosphoinositide 3-kinase (PI3K) is frequently activated in human cancer cells due to gain of function mutations in the catalytic (p110) and the regulatory (p85) subunits. The regulatory subunit consists of an SH3 domain and two SH2 domains. An oncogenic form of p85α named p65 lacking the c-terminal SH2 domain (cSH2) has been cloned from an irradiation-induced murine thymic lymphoma and transgenic mice expressing p65 in T lymphocytes develop a lymphoproliferative disorder. We have recently detected a c-terminal truncated form of p85α named p76α in a human lymphoma cell line lacking most of the cSH2 domain due to a frame shift mutation. Here, we report that the deletion of the cSH2 domain enhances the activating effects of the n-terminal SH2 domain (nSH2) mutants K379E and R340E on the PI3K/Akt pathway and micro tumor formation in a focus assay. Further analysis revealed that this transforming effect is mediated by activation of the catalytic PI3K isoform p110α and downstream signaling through mTOR. Our data further support a mechanistic model in which mutations of the cSH2 domain of p85α can abrogate its negative regulatory function on PI3K activity via the nSH2 domain of p85α. Copyright © 2012 Elsevier Inc. All rights reserved.

Identification of novel phosphatidic acid binding domain on sphingosine kinase 1 of Arabidopsis thaliana.

PubMed

Pandit, Shatakshi; Dalal, Vikram; Mishra, Girish

2018-07-01

Phosphatidic acid (PA) is an important lipid signaling molecule which interacts with Arabidopsis thaliana Sphingosine kinase1 (AtSPHK1) during several abiotic stresses particularly drought stress as a result of Abscisic acid (ABA) signaling in guard cells. PA molecules respond by generating lipid signal and/or by binding and translocating target proteins to membrane. However, site of interaction and role of PA binding to AtSPHK1 is not clear yet. Owing to the importance of AtSPHK1 during stress signaling it is imperative to decipher the site of PA interaction with AtSPHK1. To identify the PA binding region of AtSPHK1, various deletion fragments from N-terminal and C-terminal region were prepared. Results from protein lipid overlay assay using various truncated proteins of AtSPHK1 suggested the involvement of N-terminal region, between 110 and 205 amino acids, in binding with PA. In-silico analyses performed to build homologous structure of AtSPHK1 revealed that PA docking occurs in the hydrophobic cavity of DAG-Kinase domain. Deletion of amino acids 182 VSGDGI 187 perturbed PA-AtSPHK1 binding, indicating an essential role of these six amino acids in PA-AtSPHK1 binding. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Pulsed radiofrequency reduced complete Freund's adjuvant-induced mechanical hyperalgesia via the spinal c-Jun N-terminal kinase pathway.

PubMed

Chen, Kuan-Hung; Yang, Chien-Hui; Juang, Sin-Ei; Huang, Hui-Wen; Cheng, Jen-Kun; Sheen-Chen, Shyr-Ming; Cheng, Jiin-Tsuey; Lin, Chung-Ren

2014-03-01

Pulsed radiofrequency (PRF) treatment involves the pulsed application of a radiofrequency electric field to a nerve. The technology offers pain relief for patients suffering from chronic pain who do not respond well to conventional treatments. We tested whether PRF treatment attenuated complete Freund's adjuvant (CFA) induced inflammatory pain. The profile of spinal c-Jun N-terminal kinases (JNKs) phosphorylation was evaluated to elucidate the potential mechanism. Injection of CFA into the unilateral hind paw of rats induced mechanical hyperalgesia in both the ipsilateral and contralateral hind paws. We administered 500-kHz PRF treatment in 20-ms pulses, at a rate of 2 Hz (2 pulses per second) either to the sciatic nerve in the mid-thigh, or to the L4 anterior primary ramus just distal to the intervertebral foramen in both the CFA group and no-PRF group rats. Tissue samples were examined at 1, 3, 7, and 14 days following PRF treatments. Behavioral studies showed that PRF applied close to the dorsal root ganglion (DRG) significantly attenuated CFA-induced mechanical hyperalgesia compared to no-PRF group (P < .05). And western blotting revealed significant attenuation of the activation of JNK in the spinal dorsal horn compared to no-PRF group animals (P < .05). Application of PRF close to DRG provides an effective treatment for CFA-induced persistent mechanical hyperalgesia by attenuating JNK activation in the spinal dorsal horn.

Dermatophytes activate skin keratinocytes via mitogen-activated protein kinase signaling and induce immune responses.

PubMed

Achterman, Rebecca R; Moyes, David L; Thavaraj, Selvam; Smith, Adam R; Blair, Kris M; White, Theodore C; Naglik, Julian R

2015-04-01

Dermatophytes cause superficial and cutaneous fungal infections in immunocompetent hosts and invasive disease in immunocompromised hosts. However, the host mechanisms that regulate innate immune responses against these fungi are largely unknown. Here, we utilized commercially available epidermal tissues and primary keratinocytes to assess (i) damage induction by anthropophilic, geophilic, and zoophilic dermatophyte strains and (ii) the keratinocyte signaling pathways, transcription factors, and proinflammatory responses induced by a representative dermatophyte, Trichophyton equinum. Initially, five dermatophyte species were tested for their ability to invade, cause tissue damage, and induce cytokines, with Microsporum gypseum inducing the greatest level of damage and cytokine release. Using T. equinum as a representative dermatophyte, we found that the mitogen-activated protein kinase (MAPK) pathways were predominantly affected, with increased levels of phospho-p38 and phospho-Jun N-terminal protein kinase (JNK) but decreased levels of phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2). Notably, the NF-κB and PI3K pathways were largely unaffected. T. equinum also significantly increased expression of the AP-1-associated transcription factor, c-Fos, and the MAPK regulatory phosphatase, MKP1. Importantly, the ability of T. equinum to invade, cause tissue damage, activate signaling and transcription factors, and induce proinflammatory responses correlated with germination, indicating that germination may be important for dermatophyte virulence and host immune activation. Copyright © 2015, Achterman et al.

Mitogen-activated protein kinase cascades in Vitis vinifera

PubMed Central

Çakır, Birsen; Kılıçkaya, Ozan

2015-01-01

Protein phosphorylation is one of the most important mechanisms to control cellular functions in response to external and endogenous signals. Mitogen-activated protein kinases (MAPK) are universal signaling molecules in eukaryotes that mediate the intracellular transmission of extracellular signals resulting in the induction of appropriate cellular responses. MAPK cascades are composed of four protein kinase modules: MAPKKK kinases (MAPKKKKs), MAPKK kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In plants, MAPKs are activated in response to abiotic stresses, wounding, and hormones, and during plant pathogen interactions and cell division. In this report, we performed a complete inventory of MAPK cascades genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with MAPK, MAPK kinases, MAPK kinase kinases and MAPK kinase kinase kinase kinase members of Arabidopsis thaliana, we revealed the existence of 14 MAPKs, 5 MAPKKs, 62 MAPKKKs, and 7 MAPKKKKs in Vitis vinifera. We identified orthologs of V. vinifera putative MAPKs in different species, and ESTs corresponding to members of MAPK cascades in various tissues. This work represents the first complete inventory of MAPK cascades in V. vinifera and could help elucidate the biological and physiological functions of these proteins in V. vinifera. PMID:26257761

Cardiac-specific overexpression of insulin-like growth factor I (IGF-1) rescues lipopolysaccharide-induced cardiac dysfunction and activation of stress signaling in murine cardiomyocytes.

PubMed

Zhao, Peng; Turdi, Subat; Dong, Feng; Xiao, Xiaoyan; Su, Guohai; Zhu, Xinglei; Scott, Glenda I; Ren, Jun

2009-07-01

Lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, plays a key role in cardiac dysfunction in sepsis. Low circulating levels of insulin-like growth factor 1 (IGF-1) are found in sepsis, although the influence of IGF-1 on septic cardiac defect is unknown. This study was designed to examine the impact of IGF-1 on LPS-induced cardiac contractile and intracellular Ca2+ dysfunction, activation of stress signal and endoplasmic reticulum (ER) stress. Mechanical and intracellular Ca2+ properties were examined in cardiomyocytes from Fast Violet B and cardiac-specific IGF-1 overexpression mice treated with or without LPS (4 mg kg(-1), 6 h). Reactive oxygen species (ROS), protein carbonyl formation and apoptosis were measured. Activation of mitogen-activated protein kinase pathways (p38, c-jun N-terminal kinase [JNK] and extracellular signal-related kinase [ERK]), ER stress and apoptotic markers were evaluated using Western blot analysis. Our results revealed decreased peak shortening and maximal velocity of shortening/relengthening and prolonged duration of relengthening in LPS-treated Fast Violet B cardiomyocytes associated with reduced intracellular Ca2+ decay. Accumulation of ROS protein carbonyl and apoptosis were elevated after LPS treatment. Western blot analysis revealed activated p38 and JNK, up-regulated Bax, and the ER stress markers GRP78 and Gadd153 in LPS-treated mouse hearts without any change in ERK and Bcl-2. Total protein expression of p38, JNK, and ERK was unaffected by either LPS or IGF-1. Interestingly, these LPS-induced changes in mechanical and intracellular Ca2+ properties, ROS, protein carbonyl, apoptosis, stress signal activation, and ER stress markers were effectively ablated by IGF-1. In vitro LPS exposure (1 microg mL(-1)) produced cardiomyocyte mechanical dysfunction reminiscent of the in vivo setting, which was alleviated by exogenous IGF-1 (50 nM). These data collectively suggested a beneficial of IGF-1 in

Abl N-terminal cap stabilization of SH3 domain dynamics.

PubMed

Chen, Shugui; Dumitrescu, Teodora Pene; Smithgall, Thomas E; Engen, John R

2008-05-27

Crystal structures and other biochemical data indicate that the N-terminal cap (NCap) region of the Abelson tyrosine kinase (c-Abl) is important for maintaining the downregulated conformation of the kinase domain. The exact contributions that the NCap makes in stabilizing the various intramolecular interactions within c-Abl are less clear. While the NCap appears to be important for locking the SH3 and SH2 domains to the back of the kinase domain, there may be other more subtle elements of regulation. Hydrogen exchange (HX) and mass spectrometry (MS) were used to determine if the NCap contributes to intramolecular interactions involving the Abl SH3 domain. Under physiological conditions, the Abl SH3 domain underwent partial unfolding and its unfolding half-life was slowed during binding to the SH2 kinase linker, providing a unique assay for testing NCap-induced stabilization of the SH3 domain in various constructs. The results showed that the NCap stabilizes the dynamics of the SH3 domain in certain constructs but does not increase the relative affinity of the SH3 domain for the native SH2 kinase linker. The stabilization effect was absent in constructs of just the NCap and SH3 but was obvious when the SH2 domain and the SH2 kinase linker were present. These results suggest that interactions between the NCap and the SH3 domain can contribute to c-Abl stabilization in constructs that contain at least the SH2 domain, an effect that may partially compensate for the absence of the negative regulatory C-terminal tail found in the related Src family of kinases.

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

PubMed

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

2002-02-01

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

Influenza A Virus Virulence Depends on Two Amino Acids in the N-Terminal Domain of Its NS1 Protein To Facilitate Inhibition of the RNA-Dependent Protein Kinase PKR

PubMed Central

Schierhorn, Kristina L.; Jolmes, Fabian; Bespalowa, Julia; Saenger, Sandra; Peteranderl, Christin; Dzieciolowski, Julia; Mielke, Maja; Budt, Matthias; Pleschka, Stephan; Herrmann, Andreas; Herold, Susanne

2017-01-01

ABSTRACT The RNA-dependent protein kinase (PKR) has broad antiviral activity inducing translational shutdown of viral and cellular genes and is therefore targeted by various viral proteins to facilitate pathogen propagation. The pleiotropic NS1 protein of influenza A virus acts as silencer of PKR activation and ensures high-level viral replication and virulence. However, the exact manner of this inhibition remains controversial. To elucidate the structural requirements within the NS1 protein for PKR inhibition, we generated a set of mutant viruses, identifying highly conserved arginine residues 35 and 46 within the NS1 N terminus as being most critical not only for binding to and blocking activation of PKR but also for efficient virus propagation. Biochemical and Förster resonance energy transfer (FRET)-based interaction studies showed that mutation of R35 or R46 allowed formation of NS1 dimers but eliminated any detectable binding to PKR as well as to double-stranded RNA (dsRNA). Using in vitro and in vivo approaches to phenotypic restoration, we demonstrated the essential role of the NS1 N terminus for blocking PKR. The strong attenuation conferred by NS1 mutation R35A or R46A was substantially alleviated by stable knockdown of PKR in human cells. Intriguingly, both NS1 mutant viruses did not trigger any signs of disease in PKR+/+ mice, but replicated to high titers in lungs of PKR−/− mice and caused lethal infections. These data not only establish the NS1 N terminus as highly critical for neutralization of PKR's antiviral activity but also identify this blockade as an indispensable contribution of NS1 to the viral life cycle. IMPORTANCE Influenza A virus inhibits activation of the RNA-dependent protein kinase (PKR) by means of its nonstructural NS1 protein, but the underlying mode of inhibition is debated. Using mutational analysis, we identified arginine residues 35 and 46 within the N-terminal NS1 domain as highly critical for binding to and functional

The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation

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

Du, Kuo; Williams, C. David; McGill, Mitchell R.

2013-12-15

Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented whenmore » animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4–6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions. - Highlights: • 2-APB protected against APAP-induced liver injury in mice in vivo and in vitro • 2-APB protected by inhibiting APAP metabolic activation and JNK signaling pathway • DMSO inhibited APAP metabolic activation as the solvent of 2

p21-activated kinase signaling in breast cancer.

PubMed

Gururaj, Anupama E; Rayala, Suresh K; Kumar, Rakesh

2005-01-01

The p21-activated kinases signal through a number of cellular pathways fundamental to growth, differentiation and apoptosis. A wealth of information has accumulated at an impressive pace in the recent past, both with regard to previously identified targets for p21-activated kinases that regulate the actin cytoskeleton and cellular stress pathways and with regard to newly identified targets and their role in cancer. Emerging data also provide new clues towards a previously unappreciated link between these various cellular processes. The present review attempts to provide a quick tutorial to the reader about the evolving significance of p21-activated kinases and small GTPases in breast cancer, using information from mouse models, tissue culture studies, and human materials.

Her4 and Her2/neu tyrosine kinase domains dimerize and activate in a reconstituted in vitro system.

PubMed

Monsey, John; Shen, Wei; Schlesinger, Paul; Bose, Ron

2010-03-05

Her4 (ErbB-4) and Her2/neu (ErbB-2) are receptor-tyrosine kinases belonging to the epidermal growth factor receptor (EGFR) family. Crystal structures of EGFR and Her4 kinase domains demonstrate kinase dimerization and activation through an allosteric mechanism. The kinase domains form an asymmetric dimer, where the C-lobe surface of one monomer contacts the N-lobe of the other monomer. EGFR kinase dimerization and activation in vitro was previously reported using a nickel-chelating lipid-liposome system, and we now apply this system to all other members of the EGFR family. Polyhistidine-tagged Her4, Her2/neu, and Her3 kinase domains are bound to these nickel-liposomes and are brought to high local concentration, mimicking what happens to full-length receptors in vivo following ligand binding. Addition of nickel-liposomes to Her4 kinase domain results in 40-fold activation in kinase activity and marked enhancement of C-terminal tail autophosphorylation. Activation of Her4 shows a sigmoidal dependence on kinase concentration, consistent with a cooperative process requiring kinase dimerization. Her2/neu kinase activity is also activated by nickel-liposomes, and is increased further by heterodimerization with Her3 or Her4. The ability of Her3 and Her4 to heterodimerize and activate other family members is studied in vitro. Her3 kinase domain readily activates Her2/neu but is a poor activator of Her4, which differs from the prediction made by the asymmetric dimer model. Mutation of Her3 residues (952)ENI(954) to the corresponding sequence in Her4 enhanced the ability of Her3 to activate Her4, demonstrating that sequence differences on the C-lobe surface influence the heterodimerization and activation of ErbB kinase domains.

Subcellular distributions of rat CaM kinase phosphatase N and other members of the CaM kinase regulatory system.

PubMed

Kitani, Takako; Okuno, Sachiko; Takeuchi, Masayuki; Fujisawa, Hitoshi

2003-07-01

Ca2+/Calmodulin-dependent protein kinase (CaM kinase) regulatory system is composed of multifunctional CaM kinases such as CaM kinases IV and I, upstream CaM kinases such as CaM kinase kinases alpha and beta, which activate multifunctional CaM kinases, and CaM kinase phosphatases such as CaM kinase phosphatase and CaM kinase phosphatase N, which deactivate the activated multifunctional CaM kinases. To understand the combinations of CaM kinases I and IV, CaM kinase kinases alpha and beta, and CaM kinase phosphatases, the locations of the enzymes in the cell were examined by immunocytochemical studies of cultured cells. The results indicate that CaM kinase I, CaM kinase kinase beta, and CaM kinase phosphatase occur in the cytoplasm and that CaM kinase IV, CaM kinase kinase alpha (and CaM kinase kinase beta in some cell types and tissues), and CaM kinase phosphatase N occur inside the cellular nucleus, suggesting that there are at least two different sets of CaM kinase regulatory systems, one consisting of CaM kinase I, CaM kinase kinase beta, and CaM kinase phosphatase in the cytoplasm and the other consisting of CaM kinase IV, CaM kinase kinase alpha (and CaM kinase kinase beta in some cell types and tissues), and CaM kinase phosphatase N in the nucleus.

n-Propyl gallate suppresses lipopolysaccharide-induced inducible nitric oxide synthase activation through protein kinase Cδ-mediated up-regulation of heme oxygenase-1 in RAW264.7 macrophages.

PubMed

Jeon, Wookwang; Park, Seong Ji; Kim, Byung-Chul

2017-04-15

n-Propyl gallate is a synthetic phenolic antioxidant with potential anti-inflammatory effects. However, the underlying mechanism remains largely unknown. In the present study, we showed that n-propyl gallate increases the expression and activity of the heme oxygenase-1 (HO-1), a stress-inducible protein with potent anti-inflammatory activity, in RAW264.7 macrophages. The inhibition of the HO-1 activity by treatment with zinc (II) protoporphyrin IX (ZnPP) or by knockdown of the HO-1 expression with small interference RNA significantly reversed the inhibitory effect of n-Propyl gallate on activations of nuclear factor-κB (NF-κB) and inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide (LPS). An additional mechanism study using inhibitors of signaling kinases revealed the involvement of protein kinase Cδ (PKCδ) in the expression of HO-1 induced by n-Propyl gallate. Consistent with these results, n-Propyl gallate increased the intracellular levels of phosphorylated PKCδ in concentration- and time-dependent manners. The inhibitory effects of n-Propyl gallate on LPS-induced iNOS expression and nitric oxide production were also significantly attenuated by pretreatment with the PKCδ inhibitor, rottlerin, or by transfection with PKCδ (K376R), a kinase-inactive form of PKCδ. Taken together, these findings provide the first evidence that n-Propyl gallate exerts its anti-inflammatory effect through PKCδ-mediated up-regulation of HO-1 in macrophages. Copyright © 2017 Elsevier B.V. All rights reserved.

Inhibitor of apoptosis signal-regulating kinase 1 protects against acetaminophen-induced liver injury

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

Xie, Yuchao; Ramachandran, Anup; Breckenridge, David G.

Metabolic activation and oxidant stress are key events in the pathophysiology of acetaminophen (APAP) hepatotoxicity. The initial mitochondrial oxidative stress triggered by protein adduct formation is amplified by c-jun-N-terminal kinase (JNK), resulting in mitochondrial dysfunction and ultimately cell necrosis. Apoptosis signal-regulating kinase 1 (ASK1) is considered the link between oxidant stress and JNK activation. The objective of the current study was to assess the efficacy and mechanism of action of the small-molecule ASK1 inhibitor GS-459679 in a murine model of APAP hepatotoxicity. APAP (300 mg/kg) caused extensive glutathione depletion, JNK activation and translocation to the mitochondria, oxidant stress and livermore » injury as indicated by plasma ALT activities and area of necrosis over a 24 h observation period. Pretreatment with 30 mg/kg of GS-459679 almost completely prevented JNK activation, oxidant stress and injury without affecting the metabolic activation of APAP. To evaluate the therapeutic potential of GS-459679, mice were treated with APAP and then with the inhibitor. Given 1.5 h after APAP, GS-459679 was still protective, which was paralleled by reduced JNK activation and p-JNK translocation to mitochondria. However, GS-459679 treatment was not more effective than N-acetylcysteine, and the combination of GS-459679 and N-acetylcysteine exhibited similar efficacy as N-acetylcysteine monotherapy, suggesting that GS-459769 and N-acetylcysteine affect the same pathway. Importantly, inhibition of ASK1 did not impair liver regeneration as indicated by PCNA staining. In conclusion, the ASK1 inhibitor GS-459679 protected against APAP toxicity by attenuating JNK activation and oxidant stress in mice and may have therapeutic potential for APAP overdose patients. - Highlights: • Two ASK1 inhibitors protected against acetaminophen-induced liver injury. • The ASK1 inhibitors protect when used as pre- or post-treatment. • Protection by ASK1

Double-stranded RNA-activated protein kinase is a key modulator of insulin sensitivity in physiological conditions and in obesity in mice.

PubMed

Carvalho-Filho, M A; Carvalho, B M; Oliveira, A G; Guadagnini, D; Ueno, M; Dias, M M; Tsukumo, D M; Hirabara, S M; Reis, L F; Curi, R; Carvalheira, J B C; Saad, Mario J A

2012-11-01

The molecular integration of nutrient- and pathogen-sensing pathways has become of great interest in understanding the mechanisms of insulin resistance in obesity. The double-stranded RNA-dependent protein kinase (PKR) is one candidate molecule that may provide cross talk between inflammatory and metabolic signaling. The present study was performed to determine, first, the role of PKR in modulating insulin action and glucose metabolism in physiological situations, and second, the role of PKR in insulin resistance in obese mice. We used Pkr(-/-) and Pkr(+/+) mice to investigate the role of PKR in modulating insulin sensitivity, glucose metabolism, and insulin signaling in liver, muscle, and adipose tissue in response to a high-fat diet. Our data show that in lean Pkr(-/-) mice, there is an improvement in insulin sensitivity, and in glucose tolerance, and a reduction in fasting blood glucose, probably related to a decrease in protein phosphatase 2A activity and a parallel increase in insulin-induced thymoma viral oncogene-1 (Akt) phosphorylation. PKR is activated in tissues of obese mice and can induce insulin resistance by directly binding to and inducing insulin receptor substrate (IRS)-1 serine307 phosphorylation or indirectly through modulation of c-Jun N-terminal kinase and inhibitor of κB kinase β. Pkr(-/-) mice were protected from high-fat diet-induced insulin resistance and glucose intolerance and showed improved insulin signaling associated with a reduction in c-Jun N-terminal kinase and inhibitor of κB kinase β phosphorylation in insulin-sensitive tissues. PKR may have a role in insulin sensitivity under normal physiological conditions, probably by modulating protein phosphatase 2A activity and serine-threonine kinase phosphorylation, and certainly, this kinase may represent a central mechanism for the integration of pathogen response and innate immunity with insulin action and metabolic pathways that are critical in obesity.

Structural basis for the mechanism and substrate specificity of glycocyamine kinase, a phosphagen kinase family member

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

Lim, Kap; Pullalarevu, Sadhana; Surabian, Karen Talin

2010-03-12

Glycocyamine kinase (GK), a member of the phosphagen kinase family, catalyzes the Mg{sup 2+}-dependent reversible phosphoryl group transfer of the N-phosphoryl group of phosphoglycocyamine to ADP to yield glycocyamine and ATP. This reaction helps to maintain the energy homeostasis of the cell in some multicelullar organisms that encounter high and variable energy turnover. GK from the marine worm Namalycastis sp. is heterodimeric, with two homologous polypeptide chains, {alpha} and {beta}, derived from a common pre-mRNA by mutually exclusive N-terminal alternative exons. The N-terminal exon of GK{beta} encodes a peptide that is different in sequence and is 16 amino acids longermore » than that encoded by the N-terminal exon of GK{alpha}. The crystal structures of recombinant GK{alpha}{beta} and GK{beta}{beta} from Namalycastis sp. were determined at 2.6 and 2.4 {angstrom} resolution, respectively. In addition, the structure of the GK{beta}{beta} was determined at 2.3 {angstrom} resolution in complex with a transition state analogue, Mg{sup 2+}-ADP-NO{sub 3}{sup -}-glycocyamine. Consistent with the sequence homology, the GK subunits adopt the same overall fold as that of other phosphagen kinases of known structure (the homodimeric creatine kinase (CK) and the monomeric arginine kinase (AK)). As with CK, the GK N-termini mediate the dimer interface. In both heterodimeric and homodimeric GK forms, the conformations of the two N-termini are asymmetric, and the asymmetry is different than that reported previously for the homodimeric CKs from several organisms. The entire polypeptide chains of GK{alpha}{beta} are structurally defined, and the longer N-terminus of the {beta} subunit is anchored at the dimer interface. In GK{beta}{beta} the 24 N-terminal residues of one subunit and 11 N-terminal residues of the second subunit are disordered. This observation is consistent with a proposal that the GK{alpha}{beta} amino acids involved in the interface formation were

Effects of c-Jun N-terminal kinase on Activin A/Smads signaling in PC12 cell suffered from oxygen-glucose deprivation.

PubMed

Wang, J Q; Xu, Z H; Liang, W Z; He, J T; Cui, Y; Liu, H Y; Xue, L X; Shi, W; Shao, Y K; Mang, J; Xu, Z X

2016-02-29

Activin A (Act A), a member of transforming growth factor-β (TGF-β) superfamily, is an early gene in response to cerebral ischemia. Growing evidences confirm the neuroprotective effect of Act A in ischemic injury through Act A/Smads signal activation. In this process, regulation networks are involved in modulating the outcomes of Smads signaling. Among these regulators, crosstalk between c-Jun N-terminal kinase (JNK) and Smads signaling has been found in the TGF-β induced epithelial-mesenchymal transition. However, in neural ischemia, the speculative regulation between JNK and Act A/Smads signaling pathways has not been clarified. To explore this issue, an Oxygen Glucose Deprivation (OGD) model was introduced to nerve-like PC12 cells. We found that JNK signal activation occurred at the early time of OGD injury (1 h). Act A administration suppressed JNK phosphorylation. In addition, JNK inhibition could elevate the strength of Smads signaling and attenuate neural apoptosis after OGD injury. Our results indicated a negative regulation effect of JNK on Smads signaling in ischemic injury. Taken together, JNK, as a critical site for neural apoptosis and negative regulator for Act A/Smads signaling, was presumed to be a molecular therapeutic target for ischemia.

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

PubMed Central

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

2001-01-01

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

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

PubMed

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

2001-01-01

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

Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: Essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase

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

Hien, Tran Thi; Kim, Nak Doo; Pokharel, Yuba Raj

2010-08-01

We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10 {mu}g/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase weremore » enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.« less

Low humidity environmental challenge causes barrier disruption and cornification of the mouse corneal epithelium via a c-jun N-terminal kinase 2 (JNK2) pathway.

PubMed

Pelegrino, F S A; Pflugfelder, S C; De Paiva, C S

2012-01-01

Patients with tear dysfunction often experience increased irritation symptoms when subjected to drafty and/or low humidity environmental conditions. The purpose of this study was to investigate the effects of low humidity stress (LHS) on corneal barrier function and expression of cornified envelope (CE) precursor proteins in the epithelium of C57BL/6 and c-jun N-terminal kinase 2 (JNK2) knockout (KO) mice. LHS was induced in both strains by exposure to an air draft for 15 (LHS15D) or 30 days (LHS30D) at a relative humidity <30%RH. Nonstressed (NS) mice were used as controls. Oregon-green-dextran uptake was used to measure corneal barrier function. Levels of small proline-rich protein (SPRR)-2, involucrin, occludin, and MMP-9 were evaluated by immunofluorescent staining in cornea sections. Wholemount corneas immunostained for occludin were used to measure mean apical cell area. Gelatinase activity was evaluated by in situ zymography. Expression of MMP, CE and inflammatory cytokine genes was evaluated by qPCR. C57BL/6 mice exposed to LHS15D showed corneal barrier dysfunction, decreased apical corneal epithelial cell area, higher MMP-9 expression and gelatinase activity and increased involucrin and SPRR-2 immunoreactivity in the corneal epithelium compared to NS mice. JNK2KO mice were resistant to LHS-induced corneal barrier disruption. MMP-3,-9,-13, IL-1α, IL-1β, involucrin and SPRR-2a RNA transcripts were significantly increased in C57BL/6 mice at LHS15D, while no change was noted in JNK2KO mice. LHS is capable of altering corneal barrier function, promoting pathologic alteration of the TJ complex and stimulating production of CE proteins by the corneal epithelium. Activation of the JNK2 signaling pathway contributes to corneal epithelial barrier disruption in LHS. Copyright © 2011 Elsevier Ltd. All rights reserved.

The DAF-16 FOXO Transcription Factor Regulates natc-1 to Modulate Stress Resistance in Caenorhabditis elegans, Linking Insulin/IGF-1 Signaling to Protein N-Terminal Acetylation

PubMed Central

Warnhoff, Kurt; Murphy, John T.; Kumar, Sandeep; Schneider, Daniel L.; Peterson, Michelle; Hsu, Simon; Guthrie, James; Robertson, J. David; Kornfeld, Kerry

2014-01-01

The insulin/IGF-1 signaling pathway plays a critical role in stress resistance and longevity, but the mechanisms are not fully characterized. To identify genes that mediate stress resistance, we screened for C. elegans mutants that can tolerate high levels of dietary zinc. We identified natc-1, which encodes an evolutionarily conserved subunit of the N-terminal acetyltransferase C (NAT) complex. N-terminal acetylation is a widespread modification of eukaryotic proteins; however, relatively little is known about the biological functions of NATs. We demonstrated that loss-of-function mutations in natc-1 cause resistance to a broad-spectrum of physiologic stressors, including multiple metals, heat, and oxidation. The C. elegans FOXO transcription factor DAF-16 is a critical target of the insulin/IGF-1 signaling pathway that mediates stress resistance, and DAF-16 is predicted to directly bind the natc-1 promoter. To characterize the regulation of natc-1 by DAF-16 and the function of natc-1 in insulin/IGF-1 signaling, we analyzed molecular and genetic interactions with key components of the insulin/IGF-1 pathway. natc-1 mRNA levels were repressed by DAF-16 activity, indicating natc-1 is a physiological target of DAF-16. Genetic studies suggested that natc-1 functions downstream of daf-16 to mediate stress resistance and dauer formation. Based on these findings, we hypothesize that natc-1 is directly regulated by the DAF-16 transcription factor, and natc-1 is a physiologically significant effector of the insulin/IGF-1 signaling pathway that mediates stress resistance and dauer formation. These studies identify a novel biological function for natc-1 as a modulator of stress resistance and dauer formation and define a functionally significant downstream effector of the insulin/IGF-1 signaling pathway. Protein N-terminal acetylation mediated by the NatC complex may play an evolutionarily conserved role in regulating stress resistance. PMID:25330323

Role of inhibitory κB kinase and c-Jun NH2-terminal kinase in the development of hepatic insulin resistance in critical illness diabetes.

PubMed

Jiang, Shaoning; Messina, Joseph L

2011-09-01

Hyperglycemia and insulin resistance induced by acute injuries or critical illness are associated with increased mortality and morbidity, as well as later development of type 2 diabetes. The molecular mechanisms underlying the acute onset of insulin resistance following critical illness remain poorly understood. In the present studies, the roles of serine kinases, inhibitory κB kinase (IKK) and c-Jun NH(2)-terminal kinase (JNK), in the acute development of hepatic insulin resistance were investigated. In our animal model of critical illness diabetes, activation of hepatic IKK and JNK was observed as early as 15 min, concomitant with the rapid impairment of hepatic insulin signaling and increased serine phosphorylation of insulin receptor substrate 1. Inhibition of IKKα or IKKβ, or both, by adenovirus vector-mediated expression of dominant-negative IKKα or IKKβ in liver partially restored insulin signaling. Similarly, inhibition of JNK1 kinase by expression of dominant-negative JNK1 also resulted in improved hepatic insulin signaling, indicating that IKK and JNK1 kinases contribute to critical illness-induced insulin resistance in liver.

Role of inhibitory κB kinase and c-Jun NH2-terminal kinase in the development of hepatic insulin resistance in critical illness diabetes

PubMed Central

Jiang, Shaoning

2011-01-01

Hyperglycemia and insulin resistance induced by acute injuries or critical illness are associated with increased mortality and morbidity, as well as later development of type 2 diabetes. The molecular mechanisms underlying the acute onset of insulin resistance following critical illness remain poorly understood. In the present studies, the roles of serine kinases, inhibitory κB kinase (IKK) and c-Jun NH2-terminal kinase (JNK), in the acute development of hepatic insulin resistance were investigated. In our animal model of critical illness diabetes, activation of hepatic IKK and JNK was observed as early as 15 min, concomitant with the rapid impairment of hepatic insulin signaling and increased serine phosphorylation of insulin receptor substrate 1. Inhibition of IKKα or IKKβ, or both, by adenovirus vector-mediated expression of dominant-negative IKKα or IKKβ in liver partially restored insulin signaling. Similarly, inhibition of JNK1 kinase by expression of dominant-negative JNK1 also resulted in improved hepatic insulin signaling, indicating that IKK and JNK1 kinases contribute to critical illness-induced insulin resistance in liver. PMID:21680774

Effect of angiotensin II type 2 receptor on tyrosine kinase Pyk2 and c-Jun NH2-terminal kinase via SHP-1 tyrosine phosphatase activity: evidence from vascular-targeted transgenic mice of AT2 receptor.

PubMed

Matsubara, H; Shibasaki, Y; Okigaki, M; Mori, Y; Masaki, H; Kosaki, A; Tsutsumi, Y; Uchiyama, Y; Fujiyama, S; Nose, A; Iba, O; Tateishi, E; Hasegawa, T; Horiuchi, M; Nahmias, C; Iwasaka, T

2001-04-20

Angiotensin II (Ang II) has two major receptor isoforms, AT1 and AT2. AT1 transphosphorylates Ca(2+)-sensitive tyrosine kinase Pyk2 to activate c-Jun NH2-terminal kinase (JNK). Although AT2 inactivates extracellular signal-regulated kinase (ERK) via tyrosine phosphatases (PTP), the action of AT2 on Pyk2 and JNK remains undefined. Using AT2-overexpressing vascular smooth muscle cells (AT2-VSMC) from AT2-transgenic mice, we studied these undefined actions of AT2. AT1-mediated JNK activity was increased 2.2-fold by AT2 inhibition, which was abolished by orthovanadate. AT2 did not affect AT1-mediated Pyk2 phosphorylation, but attenuated c-Jun mRNA accumulation by 32%. The activity of src-homology 2 domain-containing PTP (SHP-1) was significantly upregulated 1 min after AT2 stimulation. Stable overexpression of SHP-1 dominant negative mutant in AT2-VSMC completely abolished AT2-mediated inhibition of JNK activation and c-Jun expression. These findings suggest that AT2 inhibits JNK activity by affecting the downstream signal of Pyk2 in a SHP-1-dependent manner, leading to a decrease in c-Jun expression. Copyright 2001 Academic Press.

Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis.

PubMed

Labbe, Benjamin D; Kristich, Christopher J

2017-11-01

Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes Such PASTA kinases regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and are essential for viability in certain organisms. Based on in vitro studies with purified extracellular and intracellular fragments of PASTA kinases, a model for signaling has been proposed, in which the extracellular PASTA domains bind currently undefined ligands (typically thought to be peptidoglycan, or fragments thereof) to drive kinase dimerization, which leads to enhanced kinase autophosphorylation and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo Enterococcus faecalis is a Gram-positive intestinal commensal and major antibiotic-resistant opportunistic pathogen. In E. faecalis , the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, suggesting that such antimicrobials may trigger IreK signaling. Here we show that IreK responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires both the extracellular PASTA domains and specific phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. In addition, we show that IreK responds to a signal associated with growth and/or cell division, in the absence of cell wall-active antimicrobials. Surprisingly, the ability of IreK to respond to growth and/or division does not require the extracellular PASTA domains, suggesting that IreK monitors multiple parameters for sensory input in vivo IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes and regulate critical processes. The

Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis

PubMed Central

Labbe, Benjamin D.

2017-01-01

ABSTRACT Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes. Such PASTA kinases regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and are essential for viability in certain organisms. Based on in vitro studies with purified extracellular and intracellular fragments of PASTA kinases, a model for signaling has been proposed, in which the extracellular PASTA domains bind currently undefined ligands (typically thought to be peptidoglycan, or fragments thereof) to drive kinase dimerization, which leads to enhanced kinase autophosphorylation and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo. Enterococcus faecalis is a Gram-positive intestinal commensal and major antibiotic-resistant opportunistic pathogen. In E. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, suggesting that such antimicrobials may trigger IreK signaling. Here we show that IreK responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires both the extracellular PASTA domains and specific phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. In addition, we show that IreK responds to a signal associated with growth and/or cell division, in the absence of cell wall-active antimicrobials. Surprisingly, the ability of IreK to respond to growth and/or division does not require the extracellular PASTA domains, suggesting that IreK monitors multiple parameters for sensory input in vivo. IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes and regulate critical

Astrocyte Resilience to Oxidative Stress Induced by Insulin-like Growth Factor I (IGF-I) Involves Preserved AKT (Protein Kinase B) Activity*

PubMed Central

Dávila, David; Fernández, Silvia; Torres-Alemán, Ignacio

2016-01-01

Disruption of insulin-like growth factor I (IGF-I) signaling is a key step in the development of cancer or neurodegeneration. For example, interference of the prosurvival IGF-I/AKT/FOXO3 pathway by redox activation of the stress kinases p38 and JNK is instrumental in neuronal death by oxidative stress. However, in astrocytes, IGF-I retains its protective action against oxidative stress. The molecular mechanisms underlying this cell-specific protection remain obscure but may be relevant to unveil new ways to combat IGF-I/insulin resistance. Here, we describe that, in astrocytes exposed to oxidative stress by hydrogen peroxide (H2O2), p38 activation did not inhibit AKT (protein kinase B) activation by IGF-I, which is in contrast to our previous observations in neurons. Rather, stimulation of AKT by IGF-I was significantly higher and more sustained in astrocytes than in neurons either under normal or oxidative conditions. This may be explained by phosphorylation of the phosphatase PTEN at the plasma membrane in response to IGF-I, inducing its cytosolic translocation and preserving in this way AKT activity. Stimulation of AKT by IGF-I, mimicked also by a constitutively active AKT mutant, reduced oxidative stress levels and cell death in H2O2-exposed astrocytes, boosting their neuroprotective action in co-cultured neurons. These results indicate that armoring of AKT activation by IGF-I is crucial to preserve its cytoprotective effect in astrocytes and may form part of the brain defense mechanism against oxidative stress injury. PMID:26631726

Moringa oleifera fruit induce apoptosis via reactive oxygen species-dependent activation of mitogen-activated protein kinases in human melanoma A2058 cells

PubMed Central

Guon, Tae Eun; Chung, Ha Sook

2017-01-01

The present study was performed to determine the effect of Moringa oleifera fruit extract on the apoptosis of human melanoma A2058 cells. A2058 cells were treated for 72 h with Moringa oleifera fruit extract at 50–100 µg/ml, and cell viability with apoptotic changes was examined. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) was examined. It was revealed that Moringa oleifera fruit extract significantly inhibited the cell viability and promoted apoptosis of A2058 cells in a concentration-dependent manner. Moringa oleifera fruit extract-treated A2058 cells exhibited increased activities of cleaved caspase-9 and caspase-3. It also caused an enhancement of MAPK phosphorylation and ROS production. The pro-apoptotic activity of Moringa oleifera fruit extract was significantly reversed by pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125, extracellular-signal-regulated kinase (ERK) inhibitor PD98058 or ROS inhibitor N-acetyl-L-cysteine. Taken together, Moringa oleifera fruit extract is effective in inducing mitochondrial apoptosis of A2058 cells, which is mediated through induction of ROS formation, and JNK and ERK activation. Moringa oleifera fruit extract may thus have therapeutic benefits for human melanoma A2058 cells. PMID:28789398

Moringa oleifera fruit induce apoptosis via reactive oxygen species-dependent activation of mitogen-activated protein kinases in human melanoma A2058 cells.

PubMed

Guon, Tae Eun; Chung, Ha Sook

2017-08-01

The present study was performed to determine the effect of Moringa oleifera fruit extract on the apoptosis of human melanoma A2058 cells. A2058 cells were treated for 72 h with Moringa oleifera fruit extract at 50-100 µg/ml, and cell viability with apoptotic changes was examined. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) was examined. It was revealed that Moringa oleifera fruit extract significantly inhibited the cell viability and promoted apoptosis of A2058 cells in a concentration-dependent manner. Moringa oleifera fruit extract-treated A2058 cells exhibited increased activities of cleaved caspase-9 and caspase-3. It also caused an enhancement of MAPK phosphorylation and ROS production. The pro-apoptotic activity of Moringa oleifera fruit extract was significantly reversed by pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125, extracellular-signal-regulated kinase (ERK) inhibitor PD98058 or ROS inhibitor N-acetyl-L-cysteine. Taken together, Moringa oleifera fruit extract is effective in inducing mitochondrial apoptosis of A2058 cells, which is mediated through induction of ROS formation, and JNK and ERK activation. Moringa oleifera fruit extract may thus have therapeutic benefits for human melanoma A2058 cells.

N-terminal nesprin-2 variants regulate β-catenin signalling

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

Zhang, Qiuping; Minaisah, Rose-Marie; Ferraro, Elisa

2016-07-15

The spatial compartmentalisation of biochemical signalling pathways is essential for cell function. Nesprins are a multi-isomeric family of proteins that have emerged as signalling scaffolds, herein, we investigate the localisation and function of novel nesprin-2 N-terminal variants. We show that these nesprin-2 variants display cell specific distribution and reside in both the cytoplasm and nucleus. Immunofluorescence microscopy revealed that nesprin-2 N-terminal variants colocalised with β-catenin at cell-cell junctions in U2OS cells. Calcium switch assays demonstrated that nesprin-2 and β-catenin are lost from cell-cell junctions in low calcium conditions whereas emerin localisation at the NE remained unaltered, furthermore, an N-terminal fragmentmore » of nesprin-2 was sufficient for cell-cell junction localisation and interacted with β-catenin. Disruption of these N-terminal nesprin-2 variants, using siRNA depletion resulted in loss of β-catenin from cell-cell junctions, nuclear accumulation of active β-catenin and augmented β-catenin transcriptional activity. Importantly, we show that U2OS cells lack nesprin-2 giant, suggesting that the N-terminal nesprin-2 variants regulate β-catenin signalling independently of the NE. Together, these data identify N-terminal nesprin-2 variants as novel regulators of β-catenin signalling that tether β-catenin to cell-cell contacts to inhibit β-catenin transcriptional activity. - Highlights: • N-terminal nesprin-2 variants display cell specific expression patterns. • N-terminal spectrin repeats of nesprin-2 interact with β-catenin. • N-terminal nesprin-2 variants scaffold β-catenin at cell-cell junctions.. • Nesprin-2 variants play multiple roles in β-catenin signalling.« less

GsCBRLK, a calcium/calmodulin-binding receptor-like kinase, is a positive regulator of plant tolerance to salt and ABA stress.

PubMed

Yang, Liang; Ji, Wei; Zhu, Yanming; Gao, Peng; Li, Yong; Cai, Hua; Bai, Xi; Guo, Dianjing

2010-05-01

Calcium/calmodulin-dependent kinases play vital roles in protein phosphorylation in eukaryotes, yet little is known about the phosphorylation process of calcium/calmodulin-dependent protein kinase and its role in stress signal transduction in plants. A novel plant-specific calcium-dependent calmodulin-binding receptor-like kinase (GsCBRLK) has been isolated from Glycine soja. A subcellular localization study using GFP fusion protein indicated that GsCBRLK is localized in the plasma membrane. Binding assays demonstrated that calmodulin binds to GsCBRLK with an affinity of 25.9 nM in a calcium-dependent manner and the binding motif lies between amino acids 147 to169 within subdomain II of the kinase domain. GsCBRLK undergoes autophosphorylation and Myelin Basis Protein phosphorylation in the presence of calcium. It was also found that calcium/calmodulin positively regulates GsCBRLK kinase activity through direct interaction between the calmodulin-binding domain and calmodulin. So, it is likely that GsCBRLK responds to an environmental stimulus in two ways: by increasing the protein expression level and by regulating its kinase activity through the calcium/calmodulin complex. Furthermore, cold, salinity, drought, and ABA stress induce GsCBRLK gene transcripts. Over-expression of GsCBRLK in transgenic Arabidopsis resulted in enhanced plant tolerance to high salinity and ABA and increased the expression pattern of a number of stress gene markers in response to ABA and high salt. These results identify GsCBRLK as a molecular link between the stress- and ABA-induced calcium/calmodulin signal and gene expression in plant cells.

p38 mitogen-activated protein kinase (MAPK) first regulates filamentous actin at the 8-16-cell stage during preimplantation development.

PubMed

Paliga, Andrew J M; Natale, David R; Watson, Andrew J

2005-08-01

The MAPK (mitogen-activated protein kinase) superfamily of proteins consists of four separate signalling cascades: the c-Jun N-terminal kinase or stress-activated protein kinases (JNK/SAPK); the ERKs (extracellular-signal-regulated kinases); the ERK5 or big MAPK1; and the p38 MAPK group of protein kinases, all of which are highly conserved. To date, our studies have focused on defining the role of the p38 MAPK pathway during preimplantation development. p38 MAPK regulates actin filament formation through the downstream kinases MAPKAPK2/3 (MAPK-activated protein kinase 2/3) or MAPKAPK5 [PRAK (p38 regulated/activated kinase)] and subsequently through HSP25/27 (heat-shock protein 25/27). We recently reported that 2-cell-stage murine embryos treated with cytokine-suppressive anti-inflammatory drugs (CSAIDtrade mark; SB203580 and SB220025) display a reversible blockade of development at the 8-16-cell stage, indicating that p38 (MAPK) activity is required to complete murine preimplantation development. In the present study, we have investigated the stage-specific action and role of p38 MAPK in regulating filamentous actin during murine preimplantation development. Treatment of 8-cell-stage embryos with SB203580 and SB220025 (CSAIDtrade mark) resulted in a blockade of preimplantation development, loss of rhodamine phalloidin fluorescence, MK-p (phosphorylated MAPKAPK2/3), HSP-p (phosphorylated HSP25/27) and a redistribution of alpha-catenin immunofluorescence by 12 h of treatment. In contrast, treatment of 2- and 4-cell-stage embryos with CSAIDtrade mark drugs resulted in a loss of MK-p and HSP-p, but did not result in a loss of rhodamine phalloidin fluorescence. All these effects of p38 MAPK inhibition were reversed upon removal of the inhibitor, and development resumed in a delayed but normal manner to the blastocyst stage. Treatment of 8-cell embryos with PD098059 (ERK pathway inhibitor) did not affect development or fluorescence of MK-p, HSP-p or rhodamine phalloidin

Mammalian Ste20-like protein kinase 3 mediates trophoblast apoptosis in spontaneous delivery.

PubMed

Wu, Hung-Yi; Lin, Chia-Ying; Lin, Tze-Yi; Chen, Tai-Chang; Yuan, Chiun-Jye

2008-02-01

The placenta is essential in transferring gases and nutrients from the mother to the developing fetus. Trophoblast apoptosis may cause labor or other pregnancy-related disorders. This study demonstrated the essential role of Mst3, a human Ste20-like protein kinase, in the oxidative stress-induced apoptosis of trophoblasts of term placenta in normal spontaneous delivery. Oxidative stress, but not hormones released during labor such as prostaglandin E1, oxytocin or angiotensin II, induces the expression of Mst3 and apoptosis of human term placenta after elective Cesarean section without labor pain. The role of Mst3 in oxidative stress-induced apoptosis was further demonstrated in the 3A-sub-E, a human trophoblast cell line. The H2O2-induced apoptosis of 3A-sub-E cells was largely suppressed by overexpressed Mst3KR, the kinase-dead mutant or by selective knockdown of endogenous Mst3. Further studies showed that Jun N-terminal kinase (JNK) may participate in the signaling pathway of H2O2-induced apoptosis by mediating the level of Mst3. Subsequently, caspase 3 and other downstream apoptotic components may be activated by Mst3 and trigger the apoptotic process in human trophoblasts.

Casein kinase 2 promotes interaction between Rad17 and the 9-1-1 complex through constitutive phosphorylation of the C-terminal tail of human Rad17.

PubMed

Fukumoto, Yasunori; Takahashi, Kazuaki; Suzuki, Noriyuki; Ogra, Yasumitsu; Nakayama, Yuji; Yamaguchi, Naoto

2018-06-15

An interaction between the Rad17-RFC2-5 and 9-1-1 complexes is essential for ATR-Chk1 signaling, which is one of the major DNA damage checkpoints. Recently, we showed that the polyanionic C-terminal tail of human Rad17 and the embedded conserved sequence iVERGE are important for the interaction with 9-1-1 complex. Here, we show that Rad17-S667 in the C-terminal tail is constitutively phosphorylated in vivo in a casein kinase 2-dependent manner, and the phosphorylation is important for 9-1-1 interaction. The serine phosphorylation of Rad17 could be seen in the absence of exogenous genotoxic stress, and was mostly abolished by S667A substitution. Rad17-S667 was also phosphorylated when the C-terminal tail was fused with EGFP, but the phosphorylation was inhibited by two casein kinase 2 inhibitors. Furthermore, interaction between Rad17 and the 9-1-1 complex was inhibited by the casein kinase 2 inhibitor CX-4945/Silmitasertib, and the effect was dependent on the Rad17-S667 residue, indicating that S667 phosphorylation is the only role of casein kinase 2 in the 9-1-1 interaction. Our data raise the possibility that the C-terminal tail of vertebrate Rad17 regulates ATR-Chk1 signaling through multi-site phosphorylation in the iVERGE. Copyright © 2018 Elsevier Inc. All rights reserved.

Uncaria rhynchophylla inhibits the production of nitric oxide and interleukin-1β through blocking nuclear factor κB, Akt, and mitogen-activated protein kinase activation in macrophages.

PubMed

Kim, Ji-Hee; Bae, Chang Hwan; Park, Sun Young; Lee, Sang Joon; Kim, YoungHee

2010-10-01

The stems with hook of Uncaria rhynchophylla have been used in traditional medicine as an antipyretic, antihypertensive, and anticonvulsant in China and Korea. In this study, we investigated the mechanism responsible for anti-inflammatory effects of U. rhynchophylla in RAW 264.7 macrophages. The aqueous extract of U. rhynchophylla inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and interleukin (IL)-1β secretion as well as inducible NO synthase (iNOS) expression, without affecting cell viability. Furthermore, U. rhynchophylla suppressed LPS-induced nuclear factor κB (NF-κB) activation, phosphorylation, and degradation of inhibitory protein IκB (IκB)-α, phosphorylation of Akt, extracellular signal-regulated kinase 1/2, p38 kinase, and c-Jun N-terminal kinase. These results suggest that U. rhynchophylla has the inhibitory effects on LPS-induced NO and IL-1β production in macrophages through blockade in the phosphorylation of Akt and mitogen-activated protein kinases, following IκB-α degradation and NF-κB activation.

Potential involvement of N-terminal acetylation in the quantitative regulation of the ε subunit of chloroplast ATP synthase under drought stress.

PubMed

Hoshiyasu, Saki; Kohzuma, Kaori; Yoshida, Kazuo; Fujiwara, Masayuki; Fukao, Yoichiro; Yokota, Akiho; Akashi, Kinya

2013-01-01

In plants, modulation of photosynthetic energy conversion in varying environments is often accompanied by adjustment of the abundance of photosynthetic components. In wild watermelon (Citrullus lanatus L.), proteome analysis revealed that the ε subunit of chloroplast ATP synthase occurs as two distinct isoforms with largely-different isoelectric points, although encoded by a single gene. Mass spectrometry (MS) analysis of the ε isoforms indicated that the structural difference between the ε isoforms lies in the presence or absence of an acetyl group at the N-terminus. The protein level of the non-acetylated ε isoform preferentially decreased in drought, whereas the abundance of the acetylated ε isoform was unchanged. Moreover, metalloprotease activity that decomposed the ε subunit was detected in a leaf extract from drought-stressed plants. Furthermore, in vitro assay suggested that the non-acetylated ε subunit was more susceptible to degradation by metalloaminopeptidase. We propose a model in which quantitative regulation of the ε subunit involves N-terminal acetylation and stress-induced proteases.

Effects of heat stress on serum insulin, adipokines, AMP-activated protein kinase, and heat shock signal molecules in dairy cows.

PubMed

Min, Li; Cheng, Jian-bo; Shi, Bao-lu; Yang, Hong-jian; Zheng, Nan; Wang, Jia-qi

2015-06-01

Heat stress affects feed intake, milk production, and endocrine status in dairy cows. The temperature-humidity index (THI) is employed as an index to evaluate the degree of heat stress in dairy cows. However, it is difficult to ascertain whether THI is the most appropriate measurement of heat stress in dairy cows. This experiment was conducted to investigate the effects of heat stress on serum insulin, adipokines (leptin and adiponectin), AMP-activated protein kinase (AMPK), and heat shock signal molecules (heat shock transcription factor (HSF) and heat shock proteins (HSP)) in dairy cows and to research biomarkers to be used for better understanding the meaning of THI as a bioclimatic index. To achieve these objectives, two experiments were performed. The first experiment: eighteen lactating Holstein dairy cows were used. The treatments were: heat stress (HS, THI average=81.7, n=9) and cooling (CL, THI average=53.4, n=9). Samples of HS were obtained on August 16, 2013, and samples of CL were collected on April 7, 2014 in natural conditions. The second experiment: HS treatment cows (n=9) from the first experiment were fed for 8 weeks from August 16, 2013 to October 12, 2013. Samples for moderate heat stress, mild heat stress, and no heat stress were obtained, respectively, according to the physical alterations of the THI. Results showed that heat stress significantly increased the serum adiponectin, AMPK, HSF, HSP27, HSP70, and HSP90 (P<0.05). Adiponectin is strongly associated with AMPK. The increases of adiponectin and AMPK may be one of the mechanisms to maintain homeostasis in heat-stressed dairy cows. When heat stress treatment lasted 8 weeks, a higher expression of HSF and HSP70 was observed under moderate heat stress. Serum HSF and HSP70 are sensitive and accurate in heat stress and they could be potential indicators of animal response to heat stress. We recommend serum HSF and HSP70 as meaningful biomarkers to supplement the THI and evaluate moderate heat

Effects of heat stress on serum insulin, adipokines, AMP-activated protein kinase, and heat shock signal molecules in dairy cows*

PubMed Central

Min, Li; Cheng, Jian-bo; Shi, Bao-lu; Yang, Hong-jian; Zheng, Nan; Wang, Jia-qi

2015-01-01

Heat stress affects feed intake, milk production, and endocrine status in dairy cows. The temperature-humidity index (THI) is employed as an index to evaluate the degree of heat stress in dairy cows. However, it is difficult to ascertain whether THI is the most appropriate measurement of heat stress in dairy cows. This experiment was conducted to investigate the effects of heat stress on serum insulin, adipokines (leptin and adiponectin), AMP-activated protein kinase (AMPK), and heat shock signal molecules (heat shock transcription factor (HSF) and heat shock proteins (HSP)) in dairy cows and to research biomarkers to be used for better understanding the meaning of THI as a bioclimatic index. To achieve these objectives, two experiments were performed. The first experiment: eighteen lactating Holstein dairy cows were used. The treatments were: heat stress (HS, THI average=81.7, n=9) and cooling (CL, THI average=53.4, n=9). Samples of HS were obtained on August 16, 2013, and samples of CL were collected on April 7, 2014 in natural conditions. The second experiment: HS treatment cows (n=9) from the first experiment were fed for 8 weeks from August 16, 2013 to October 12, 2013. Samples for moderate heat stress, mild heat stress, and no heat stress were obtained, respectively, according to the physical alterations of the THI. Results showed that heat stress significantly increased the serum adiponectin, AMPK, HSF, HSP27, HSP70, and HSP90 (P<0.05). Adiponectin is strongly associated with AMPK. The increases of adiponectin and AMPK may be one of the mechanisms to maintain homeostasis in heat-stressed dairy cows. When heat stress treatment lasted 8 weeks, a higher expression of HSF and HSP70 was observed under moderate heat stress. Serum HSF and HSP70 are sensitive and accurate in heat stress and they could be potential indicators of animal response to heat stress. We recommend serum HSF and HSP70 as meaningful biomarkers to supplement the THI and evaluate moderate heat

Protein kinases: mechanisms and downstream targets in inflammation-mediated obesity and insulin resistance.

PubMed

Nandipati, Kalyana C; Subramanian, Saravanan; Agrawal, Devendra K

2017-02-01

Obesity-induced low-grade inflammation (metaflammation) impairs insulin receptor signaling. This has been implicated in the development of insulin resistance. Insulin signaling in the target tissues is mediated by stress kinases such as p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, inhibitor of NF-kB kinase complex β (IKKβ), AMP-activated protein kinase, protein kinase C, Rho-associated coiled-coil containing protein kinase, and RNA-activated protein kinase. Most of these kinases phosphorylate several key regulators in glucose homeostasis. The phosphorylation of serine residues in the insulin receptor and IRS-1 molecule results in diminished enzymatic activity in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. This has been one of the key mechanisms observed in the tissues that are implicated in insulin resistance especially in type 2 diabetes mellitus (T2-DM). Identifying the specific protein kinases involved in obesity-induced chronic inflammation may help in developing the targeted drug therapies to minimize the insulin resistance. This review is focused on the protein kinases involved in the inflammatory cascade and molecular mechanisms and their downstream targets with special reference to obesity-induced T2-DM.

Hydrophobic interaction between the SH2 domain and the kinase domain is required for the activation of Csk.

PubMed

Mikkola, Esa T; Gahmberg, Carl G

2010-06-18

The protein tyrosine kinase C-terminal Src kinase (Csk) is activated by the engagement of its Src homology (SH) 2 domain. However, the molecular mechanism required for this is not completely understood. The crystal structure of the active Csk indicates that Csk could be activated by contact between the SH2 domain and the beta3-alphaC loop in the N-terminal lobe of the kinase domain. To study the importance of this interaction for the SH2-domain-mediated activation of Csk, we mutated the amino acid residues forming the contacts between the SH2 domain and the beta3-alphaC loop. The mutation of the beta3-alphaC loop Ala228 to glycine and of the SH2 domain Tyr116, Tyr133, Leu138, and Leu149 to alanine resulted in the inability of the SH2 domain ligand to activate Csk. Furthermore, the overexpressed Csk mutants A228G, Y133A/Y116A, L138A, and L149A were unable to efficiently inactivate endogenous Src in human embryonic kidney 293 cells. The results suggest that the SH2-domain-mediated activation of Csk is dependent on the binding of the beta3-alphaC loop Ala228 to the hydrophobic pocket formed by the side chains of Tyr116, Tyr133, Leu138, and Leu149 on the surface of the SH2 domain. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Nef Proteins Show Distinct Patterns and Mechanisms of Src Kinase Activation

PubMed Central

Greenway, Alison L.; Dutartre, Hélène; Allen, Kelly; McPhee, Dale A.; Olive, Daniel; Collette, Yves

1999-01-01

The nef gene from human and simian immunodeficiency viruses (HIV and SIV) regulates cell function and viral replication, possibly through binding of the nef product to cellular proteins, including Src family tyrosine kinases. We show here that the Nef protein encoded by SIVmac239 interacts with and also activates the human Src kinases Lck and Hck. This is in direct contrast to the inhibitory effect of HIV type 1 (HIV-1) Nef on Lck catalytic activity. Unexpectedly, however, the interaction of SIV Nef with human Lck or Hck is not mediated via its consensus proline motif, which is known to mediate HIV-1 Nef binding to Src homology 3 (SH3) domains, and various experimental analyses failed to show significant interaction of SIV Nef with the SH3 domain of either kinase. Instead, SIV Nef can bind Lck and Hck SH2 domains, and its N-terminal 50 amino acid residues are sufficient for Src kinase binding and activation. Our results provide evidence for multiple mechanisms by which Nef binds to and regulates Src kinases. PMID:10364375

Inhibition of adrenergic human prostate smooth muscle contraction by the inhibitors of c-Jun N-terminal kinase, SP600125 and BI-78D3.

PubMed

Strittmatter, F; Walther, S; Gratzke, C; Göttinger, J; Beckmann, C; Roosen, A; Schlenker, B; Hedlund, P; Andersson, K E; Stief, C G; Hennenberg, M

2012-07-01

BACKGROUND AND PURPOSE α(1) -Adrenoceptor-induced contraction of prostate smooth muscle is mediated by calcium- and Rho kinase-dependent mechanisms. In addition, other mechanisms, such as activation of c-jun N-terminal kinase (JNK) may be involved. Here, we investigated whether JNK participates in α(1)-adrenoceptor-induced contraction of human prostate smooth muscle. EXPERIMENTAL APPROACH Prostate tissue was obtained from patients undergoing radical prostatectomy. Effects of the JNK inhibitors SP600125 (50 µM) and BI-78D3 (30 µM) on contractions induced by phenylephrine, noradrenaline and electric field stimulation (EFS) were studied in myographic measurements. JNK activation by noradrenaline (30 µM) and phenylephrine (10 µM), and the effects of JNK inhibitors of c-Jun phosphorylation were assessed by Western blot analyses with phospho-specific antibodies. Expression of JNK was studied by immunohistochemistry and fluorescence double staining. KEY RESULTS The JNK inhibitors SP600125 and BI-78D3 reduced phenylephrine- and noradrenaline-induced contractions of human prostate strips. In addition, SP600125 reduced EFS-induced contraction of prostate strips. Stimulation of prostate tissue with noradrenaline or phenylephrine in vitro resulted in activation of JNK. Incubation of prostate tissue with SP600125 or BI-78D3 reduced the phosphorylation state of c-Jun. Immunohistochemical staining demonstrated the expression of JNK in smooth muscle cells of human prostate tissue. Fluorescence staining showed that α(1A)-adrenoceptors and JNK are expressed in the same cells. CONCLUSIONS AND IMPLICATIONS Activation of JNK is involved in α(1)-adrenoceptor-induced prostate smooth muscle contraction. Models of α(1)-adrenoceptor-mediated prostate smooth muscle contraction should include this JNK-dependent mechanism. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Inhibition of inhibitor of kappaB kinases stimulates hepatic stellate cell apoptosis and accelerated recovery from rat liver fibrosis.

PubMed

Oakley, Fiona; Meso, Muriel; Iredale, John P; Green, Karen; Marek, Carylyn J; Zhou, Xiaoying; May, Michael J; Millward-Sadler, Harry; Wright, Matthew C; Mann, Derek A

2005-01-01

Resolution of liver fibrosis is associated with clearance of hepatic myofibroblasts by apoptosis; development of strategies that promote this process in a selective way is therefore important. The aim of this study was to determine whether the inhibitor of kappaB kinase suppressor sulfasalazine stimulates hepatic myofibroblast apoptosis and recovery from fibrosis. Hepatic myofibroblasts were generated by culture activation of rat and human hepatic stellate cells. Fibrosis was established in rat livers by chronic injury with carbon tetrachloride followed by recovery with or without sulfasalazine (150 mg/kg) treatment. Treatment of hepatic stellate cells with sulfasalazine (0.5-2.0 mmol/L) induced apoptosis of activated rat and human hepatic stellate cells. A single in vivo administration of sulfasalazine promoted accelerated recovery from fibrosis as assessed by improved fibrosis score, selective clearance of smooth muscle alpha-actin-positive myofibroblasts, reduced hepatic procollagen I and tissue inhibitor of metalloproteinase 1 messenger RNA expression, and increased matrix metalloproteinase 2 activity. Mechanistic studies showed that sulfasalazine selectively blocks nuclear factor-kappaB-dependent gene transcription, inhibits hepatic stellate cell expression of Gadd45beta, stimulates phosphorylation of Jun N-terminal kinase 2, and promotes apoptosis by a mechanism that is prevented by the Jun N-terminal kinase inhibitor SP600125. As further evidence for a survival role for the inhibitor of kappaB kinase/nuclear factor-kappaB pathway in activated hepatic stellate cells, a highly selective cell-permeable peptide inhibitor of kappaB kinase activation also stimulated hepatic stellate cell apoptosis via a Jun N-terminal kinase-dependent mechanism. Inhibition of the inhibitor of kappaB kinase/nuclear factor-kappaB pathway is sufficient to increase the rate at which activated hepatic stellate cells undergo apoptosis both in vitro and in vivo, and drugs that

A novel transmembrane Ser/Thr kinase complexes with protein phosphatase-1 and inhibitor-2.

PubMed

Wang, Hong; Brautigan, David L

2002-12-20

Protein kinases and protein phosphatases exert coordinated control over many essential cellular processes. Here, we describe the cloning and characterization of a novel human transmembrane protein KPI-2 (Kinase/Phosphatase/Inhibitor-2) that was identified by yeast two-hybrid using protein phosphatase inhibitor-2 (Inh2) as bait. KPI-2 mRNA was predominantly expressed in skeletal muscle. KPI-2 is a 1503-residue protein with two predicted transmembrane helices at the N terminus, a kinase domain, followed by a C-terminal domain. The transmembrane helices were sufficient for targeting proteins to the membrane. KPI-2 kinase domain has about 60% identity with its closest relative, a tyrosine kinase. However, it only exhibited serine/threonine kinase activity in autophosphorylation reactions or with added substrates. KPI-2 kinase domain phosphorylated protein phosphatase-1 (PP1C) at Thr(320), which attenuated PP1C activity. KPI-2 C-terminal domain directly associated with PP1C, and this required a VTF motif. Inh2 associated with KPI-2 C-terminal domain with and without PP1C. Thus, KPI-2 is a kinase with sites to associate with PP1C and Inh2 to form a regulatory complex that is localized to membranes.

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

PubMed

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

2016-03-01

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

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

PubMed Central

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

2016-01-01

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

Oxidized Low-Density Lipoprotein-Activated c-Jun NH2-Terminal Kinase Regulates Manganese Superoxide Dismutase Ubiquitination

PubMed Central

Takabe, Wakako; Li, Rongsong; Ai, Lisong; Yu, Fei; Berliner, Judith A.; Hsiai, Tzung K.

2012-01-01

Objective Oxidized low-density lipoprotein (oxLDL) modulates intracellular redox status and induces apoptosis in endothelial cells. However, the signal pathways and molecular mechanism remain unknown. In this study, we investigated the role of manganese superoxide dismutase (Mn-SOD) on oxLDL-induced apoptosis via c-Jun NH2-terminal kinase (JNK)-mediated ubiquitin/proteasome pathway. Methods and Results OxLDL induced JNK phosphorylation that peaked at 30 minutes in human aortic endothelial cells. Fluorescence-activated cell sorting analysis revealed that oxLDL increased mitochondrial superoxide production by 1.88±0.19-fold and mitochondrial membrane potential by 18%. JNK small interference RNA (siJNK) reduced oxLDL-induced mitochondrial superoxide production by 88.4% and mitochondrial membrane potential by 61.7%. OxLDL did not affect Mn-SOD mRNA expression, but it significantly reduced Mn-SOD protein level, which was restored by siJNK. Immunoprecipitation by ubiquitin antibody revealed that oxLDL increased ubiquitination of Mn-SOD, which was inhibited by siJNK. OxLDL-induced caspase-3 activities were also attenuated by siJNK but were enhanced by Mn-SOD small interfering RNA. Furthermore, overexpression of Mn-SOD abrogated oxLDL-induced caspase-3 activities. Conclusion OxLDL-induced JNK activation regulates mitochondrial redox status and Mn-SOD protein degradation via JNK-dependent ubiquitination, leading to endothelial cell apoptosis. PMID:20139358

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

PubMed Central

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

1998-01-01

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

Identification of a Src kinase SH3 binding site in the C-terminal domain of the human ErbB2 receptor tyrosine kinase.

PubMed

Bornet, Olivier; Nouailler, Matthieu; Feracci, Michaël; Sebban-Kreuzer, Corinne; Byrne, Deborah; Halimi, Hubert; Morelli, Xavier; Badache, Ali; Guerlesquin, Françoise

2014-06-05

Overexpression of the ErbB2 receptor tyrosine kinase is associated with most aggressive tumors in breast cancer patients and is thus one of the main investigated therapeutic targets. Human ErbB2 C-terminal domain is an unstructured anchor that recruits specific adaptors for signaling cascades resulting in cell growth, differentiation and migration. Herein, we report the presence of a SH3 binding motif in the proline rich unfolded ErbB2 C-terminal region. NMR analysis of this motif supports a PPII helix conformation and the binding to Fyn-SH3 domain. The interaction of a kinase of the Src family with ErbB2 C-terminal domain could contribute to synergistic intracellular signaling and enhanced oncogenesis. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Disruption of the LOV-Jalpha helix interaction activates phototropin kinase activity.

PubMed

Harper, Shannon M; Christie, John M; Gardner, Kevin H

2004-12-28

Light plays a crucial role in activating phototropins, a class of plant photoreceptors that are sensitive to blue and UV-A wavelengths. Previous studies indicated that phototropin uses a bound flavin mononucleotide (FMN) within its light-oxygen-voltage (LOV) domain to generate a protein-flavin covalent bond under illumination. In the C-terminal LOV2 domain of Avena sativa phototropin 1, formation of this bond triggers a conformational change that results in unfolding of a helix external to this domain called Jalpha [Harper, S. M., et al. (2003) Science 301, 1541-1545]. Though the structural effects of illumination were characterized, it was unknown how these changes are coupled to kinase activation. To examine this, we made a series of point mutations along the Jalpha helix to disrupt its interaction with the LOV domain in a manner analogous to light activation. Using NMR spectroscopy and limited proteolysis, we demonstrate that several of these mutations displace the Jalpha helix from the LOV domain independently of illumination. When placed into the full-length phototropin protein, these point mutations display constitutive kinase activation, without illumination of the sample. These results indicate that unfolding of the Jalpha helix is the critical event in regulation of kinase signaling for the phototropin proteins.

Calcium-mediated oxidative stress: a common mechanism in tight junction disruption by different types of cellular stress.

PubMed

Gangwar, Ruchika; Meena, Avtar S; Shukla, Pradeep K; Nagaraja, Archana S; Dorniak, Piotr L; Pallikuth, Sandeep; Waters, Christopher M; Sood, Anil; Rao, RadhaKrishna

2017-02-20

The role of reactive oxygen species (ROS) in osmotic stress, dextran sulfate sodium (DSS) and cyclic stretch-induced tight junction (TJ) disruption was investigated in Caco-2 cell monolayers in vitro and restraint stress-induced barrier dysfunction in mouse colon in vivo Live cell imaging showed that osmotic stress, cyclic stretch and DSS triggered rapid production of ROS in Caco-2 cell monolayers, which was blocked by depletion of intracellular Ca 2+ by 1,2-bis-( o -aminophenoxy)ethane- N , N , N ', N '-tetraacetic acid. Knockdown of Ca V 1.3 or TRPV6 channels blocked osmotic stress and DSS-induced ROS production and attenuated TJ disruption and barrier dysfunction. N -Acetyl l-cysteine (NAC) and l- N G -Nitroarginine methyl ester (l-NAME) blocked stress-induced TJ disruption and barrier dysfunction. NAC and l-NAME also blocked stress-induced activation of c-Jun N -terminal kinase (JNK) and c-Src. ROS was colocalized with the mitochondrial marker in stressed cells. Cyclosporin A blocked osmotic stress and DSS-induced ROS production, barrier dysfunction, TJ disruption and JNK activation. Mitochondria-targeted Mito-TEMPO blocked osmotic stress and DSS-induced barrier dysfunction and TJ disruption. Chronic restraint stress in mice resulted in the elevation of intracellular Ca 2+ , activation of JNK and c-Src, and disruption of TJ in the colonic epithelium. Furthermore, corticosterone administration induced JNK and c-Src activation, TJ disruption and protein thiol oxidation in colonic mucosa. The present study demonstrates that oxidative stress is a common signal in the mechanism of TJ disruption in the intestinal epithelium by different types of cellular stress in vitro and bio behavioral stress in vivo . © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Requirement for pectin methyl esterase and preference for fragmented over native pectins for wall-associated kinase-activated, EDS1/PAD4-dependent stress response in Arabidopsis.

PubMed

Kohorn, Bruce D; Kohorn, Susan L; Saba, Nicholas J; Martinez, Victoriano Meco

2014-07-04

The wall-associated kinases (WAKs) have a cytoplasmic protein kinase domain that spans the plasma membrane and binds pectin in the extracellular matrix of plants. WAKs are required for cell expansion during Arabidopsis seedling development but are also an integral part of the response to pathogens and stress that present oligogalacturonides (OGs), which subsequently bind to WAKs and activate a MPK6 (mitogen-activated protein kinase)-dependent pathway. It was unclear how WAKs distinguish native pectin polymers and OGs to activate one or the other of these two pathways. A dominant allele of WAK2 constitutively activates the stress response, and we show here that the effect is dependent upon EDS1 and PAD4, transcriptional activators involved in the pathogen response. Moreover, the WAK2 dominant allele is suppressed by a null allele of a pectin methyl esterase (PME3) whose activity normally leads to cross-linking of pectins in the cell wall. Although OGs activate a transcriptional response in wild type, the response is enhanced in a pme3/pme3 null, consistent with a competition by OG and native polymers for activation of WAKs. This provides a plausible mechanism for WAKs to distinguish an expansion from a stress pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Requirement for Pectin Methyl Esterase and Preference for Fragmented over Native Pectins for Wall-associated Kinase-activated, EDS1/PAD4-dependent Stress Response in Arabidopsis*

PubMed Central

Kohorn, Bruce D.; Kohorn, Susan L.; Saba, Nicholas J.; Martinez, Victoriano Meco

2014-01-01

The wall-associated kinases (WAKs) have a cytoplasmic protein kinase domain that spans the plasma membrane and binds pectin in the extracellular matrix of plants. WAKs are required for cell expansion during Arabidopsis seedling development but are also an integral part of the response to pathogens and stress that present oligogalacturonides (OGs), which subsequently bind to WAKs and activate a MPK6 (mitogen-activated protein kinase)-dependent pathway. It was unclear how WAKs distinguish native pectin polymers and OGs to activate one or the other of these two pathways. A dominant allele of WAK2 constitutively activates the stress response, and we show here that the effect is dependent upon EDS1 and PAD4, transcriptional activators involved in the pathogen response. Moreover, the WAK2 dominant allele is suppressed by a null allele of a pectin methyl esterase (PME3) whose activity normally leads to cross-linking of pectins in the cell wall. Although OGs activate a transcriptional response in wild type, the response is enhanced in a pme3/pme3 null, consistent with a competition by OG and native polymers for activation of WAKs. This provides a plausible mechanism for WAKs to distinguish an expansion from a stress pathway. PMID:24855660

Involvement of Alpha-PAK-Interacting Exchange Factor in the PAK1–c-Jun NH2-Terminal Kinase 1 Activation and Apoptosis Induced by Benzo[a]pyrene

PubMed Central

Yoshii, Shigeto; Tanaka, Masamitsu; Otsuki, Yoshiro; Fujiyama, Toshiharu; Kataoka, Hideki; Arai, Hajime; Hanai, Hiroyuki; Sugimura, Haruhiko

2001-01-01

Benzo[a]pyrene [B(a)P], a potent procarcinogen found in combustion products such as diesel exhaust and cigarette smoke, has been recently shown to activate the c-Jun NH2-terminal kinase 1 (JNK1) and induce caspase-3-mediated apoptosis in Hepa1c1c7 cells. However, the molecules of the signaling pathway that control the mitogen-activated protein kinase cascades induced by B(a)P and the interaction between those and apoptosis by B(a)P have not been well defined. We report here that B(a)P promoted Cdc42/Rac1, p21-activated kinase 1 (PAK1), and JNK1 activities in 293T and HeLa cells. Moreover, alpha-PAK-interacting exchange factor (α PIX) mRNA and its protein expression were upregulated by B(a)P. While overexpression of an active mutant of α PIX (ΔCH) facilitated B(a)P-induced activation of Cdc42/Rac1, PAK1, and JNK1, overexpression of mutated αPIX (L383R, L384S), which lacks guanine nucleotide exchange factor activity, SH3 domain-deleted αPIX (Δ SH3), which lacks the ability to bind PAK, kinase-negative PAK1 (K299R), and kinase-negative SEK1 (K220A, K224L) inhibited B(a)P-triggered JNK1 activation. Interestingly, overexpression of αPIX (Δ CH) and a catalytically active mutant PAK1 (T423E) accelerated B(a)P-induced apoptosis in HeLa cells, whereas αPIX (Δ SH3), PAK1 (K299R), and SEK 1 (K220A, K224L) inhibited B(a)P-initiated apoptosis. Finally, a preferential caspase inhibitor, Z-Asp-CH2-DCB, strongly blocked the αPIX (Δ CH)-enhanced apoptosis in cells treated with B(a)P but did not block PAK1/JNK1 activation. Taken together, these results indicate that αPIX plays a crucial role in B(a)P-induced apoptosis through activation of the JNK1 pathway kinases. PMID:11564864

The metabotropic glutamate receptor activates the lipid kinase PI3K in Drosophila motor neurons through the calcium/calmodulin-dependent protein kinase II and the nonreceptor tyrosine protein kinase DFak.

PubMed

Chun-Jen Lin, Curtis; Summerville, James B; Howlett, Eric; Stern, Michael

2011-07-01

Ligand activation of the metabotropic glutamate receptor (mGluR) activates the lipid kinase PI3K in both the mammalian central nervous system and Drosophila motor nerve terminal. In several subregions of the mammalian brain, mGluR-mediated PI3K activation is essential for a form of synaptic plasticity termed long-term depression (LTD), which is implicated in neurological diseases such as fragile X and autism. In Drosophila larval motor neurons, ligand activation of DmGluRA, the sole Drosophila mGluR, similarly mediates a PI3K-dependent downregulation of neuronal activity. The mechanism by which mGluR activates PI3K remains incompletely understood in either mammals or Drosophila. Here we identify CaMKII and the nonreceptor tyrosine kinase DFak as critical intermediates in the DmGluRA-dependent activation of PI3K at Drosophila motor nerve terminals. We find that transgene-induced CaMKII inhibition or the DFak(CG1) null mutation each block the ability of glutamate application to activate PI3K in larval motor nerve terminals, whereas transgene-induced CaMKII activation increases PI3K activity in motor nerve terminals in a DFak-dependent manner, even in the absence of glutamate application. We also find that CaMKII activation induces other PI3K-dependent effects, such as increased motor axon diameter and increased synapse number at the larval neuromuscular junction. CaMKII, but not PI3K, requires DFak activity for these increases. We conclude that the activation of PI3K by DmGluRA is mediated by CaMKII and DFak.

Select human cancer mutants of NRMT1 alter its catalytic activity and decrease N-terminal trimethylation.

PubMed

Shields, Kaitlyn M; Tooley, John G; Petkowski, Janusz J; Wilkey, Daniel W; Garbett, Nichola C; Merchant, Michael L; Cheng, Alan; Schaner Tooley, Christine E

2017-08-01

A subset of B-cell lymphoma patients have dominant mutations in the histone H3 lysine 27 (H3K27) methyltransferase EZH2, which change it from a monomethylase to a trimethylase. These mutations occur in aromatic resides surrounding the active site and increase growth and alter transcription. We study the N-terminal trimethylase NRMT1 and the N-terminal monomethylase NRMT2. They are 50% identical, but differ in key aromatic residues in their active site. Given how these residues affect EZH2 activity, we tested whether they are responsible for the distinct catalytic activities of NRMT1/2. Additionally, NRMT1 acts as a tumor suppressor in breast cancer cells. Its loss promotes oncogenic phenotypes but sensitizes cells to DNA damage. Mutations of NRMT1 naturally occur in human cancers, and we tested a select group for altered activity. While directed mutation of the aromatic residues had minimal catalytic effect, NRMT1 mutants N209I (endometrial cancer) and P211S (lung cancer) displayed decreased trimethylase and increased monomethylase/dimethylase activity. Both mutations are located in the peptide-binding channel and indicate a second structural region impacting enzyme specificity. The NRMT1 mutants demonstrated a slower rate of trimethylation and a requirement for higher substrate concentration. Expression of the mutants in wild type NRMT backgrounds showed no change in N-terminal methylation levels or growth rates, demonstrating they are not acting as dominant negatives. Expression of the mutants in cells lacking endogenous NRMT1 resulted in minimal accumulation of N-terminal trimethylation, indicating homozygosity could help drive oncogenesis or serve as a marker for sensitivity to DNA damaging chemotherapeutics or γ-irradiation. © 2017 The Protein Society.

Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury.

PubMed

Yoshida, Katsunori; Matsuzaki, Koichi; Mori, Shigeo; Tahashi, Yoshiya; Yamagata, Hideo; Furukawa, Fukiko; Seki, Toshihito; Nishizawa, Mikio; Fujisawa, Junichi; Okazaki, Kazuichi

2005-04-01

After liver injury, transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) regulate the activation of hepatic stellate cells (HSCs) and tissue remodeling. Mechanisms of PDGF signaling in the TGF-beta-triggered cascade are not completely understood. TGF-beta signaling involves phosphorylation of Smad2 and Smad3 at linker and C-terminal regions. Using antibodies to distinguish Smad2/3 phosphorylated at linker regions from those phosphorylated at C-terminal regions, we investigated Smad2/3-mediated signaling in rat liver injured by CCl(4) administration and in cultured HSCs. In acute liver injury, Smad2/3 were transiently phosphorylated at both regions. Although linker-phosphorylated Smad2 remained in the cytoplasm of alpha-smooth muscle actin-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas, linker-phosphorylated Smad3 accumulated in the nuclei. c-Jun N-terminal kinase (JNK) in the activated HSCs directly phosphorylated Smad2/3 at linker regions. Co-treatment of primary cultured HSCs with TGF-beta and PDGF activated the JNK pathway, subsequently inducing endogenous linker phosphorylation of Smad2/3. The JNK pathway may be involved in migration of resident HSCs within the space of Disse to the sites of tissue damage because the JNK inhibitor SP600125 inhibited HSC migration induced by TGF-beta and PDGF signals. Moreover, treatment of HSCs with both TGF-beta and PDGF increased transcriptional activity of plasminogen activator inhibitor-1 through linker phosphorylation of Smad3. In conclusion, TGF-beta and PDGF activate HSCs by transmitting their signals through JNK-mediated Smad2/3 phosphorylation at linker regions, both in vivo and in vitro.

Alkyl isothiocyanates suppress epidermal growth factor receptor kinase activity but augment tyrosine kinase activity.

PubMed

Nomura, Takahiro; Uehara, Yoshimasa; Kawajiri, Hiroo; Ryoyama, Kazuo; Yamori, Takao; Fuke, Yoko

2009-10-01

We have reported the in vitro and in vivo anticancer activities of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) derived from a Japanese spice, wasabi. In order to obtain some clues about the mechanism of the anticancer activity, we have studied the effect of alkyl isothiocyanates (MITCs) on protein kinase activities. The anti-autophosphorylation activity of MITCs with respect to the epidermal growth factor (EGF)-stimulated receptor kinase of A431 epidermoid carcinoma cells was examined by incorporation of radioactive ATP into an acid-insoluble fraction. Their anti-phosphorylation activity with respect to the non-receptor protein kinase was analyzed by a standard SDS-PAGE method. All the tested MITCs interfered with the EGF-stimulated receptor kinase activity in a dose-dependent manner, although their effects were less than 1/10 of that of erbstatin in microg/ml. On the other hand, the MITCs did not interfere with non-receptor kinases (kinase A, kinase C, tyrosine kinase and calmodulin dependent kinase III), but enhanced non-receptor tyrosine kinase. A possible anticancer mechanism of MITCs may involve the suppression of EGF receptor kinase activity and augmentation of non-receptor PTK.

Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

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

Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas

Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 thatmore » forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.« less

Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM)-mediated Inhibitory Signaling is Regulated by Sequential Phosphorylation Mediated by Distinct Nonreceptor Tyrosine Kinases: A Case Study Involving PECAM-1

PubMed Central

Tourdot, Benjamin E.; Brenner, Michelle K.; Keough, Kathleen C.; Holyst, Trudy; Newman, Peter J.; Newman, Debra K.

2013-01-01

The activation state of many blood and vascular cells is tightly controlled by a delicate balance between receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs) and those that contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Precisely how the timing of cellular activation by ITAM-coupled receptors is regulated by ITIM-containing receptors is, however, poorly understood. Using platelet endothelial cell adhesion molecule 1 (PECAM-1) as a prototypical ITIM-bearing receptor, we demonstrate that initiation of inhibitory signaling occurs via a novel, sequential process in which Src family kinases phosphorylate the C-terminal ITIM, thereby enabling phosphorylation of the N-terminal ITIM of PECAM-1 by other Src homology 2 domain-containing non-receptor tyrosine kinases (NRTKs). NRTKs capable of mediating the second phosphorylation event include C-terminal Src kinase (Csk) and Bruton’s tyrosine kinase (Btk). Btk and Csk function downstream of phosphatidylinositol 3-kinase (PI3K) activation during ITAM-dependent platelet activation. In ITAM-activated platelets that were treated with a PI3K inhibitor, PECAM-1 was phosphorylated but did not bind the tandem SH2 domain-containing tyrosine phosphatase SHP-2, indicating that it was not phosphorylated on its N-terminal ITIM. Csk bound to and phosphorylated PECAM-1 more efficiently than did Btk, and required its SH2 domain to perform these functions. Additionally, the phosphorylation of the N-terminal ITIM of Siglec-9 by Csk is enhanced by the prior phosphorylation of its C-terminal ITIM, providing evidence that the ITIMs of other dual ITIM-containing receptors are also sequentially phosphorylated. On the basis of these findings, we propose that sequential ITIM phosphorylation provides a general mechanism for precise temporal control over the recruitment and activation of tandem SH2 domain-containing tyrosine phosphatases that dampen ITAM-dependent signals. PMID:23418871

Cloning, expression and N-terminal myristoylation of CpCPK1, a calcium-dependent protein kinase from zucchini (Cucurbita pepo L.).

PubMed

Ellard-Ivey, M; Hopkins, R B; White, T J; Lomax, T L

1999-01-01

We have isolated a full-length cDNA clone (CpCDPK1) encoding a calcium-dependent protein kinase (CDPK) gene from zucchini (Cucurbita pepo L.). The predicted amino acid sequence of the cDNA shows a remarkably high degree of similarity to members of the CDPK gene family from Arabidopsis thaliana, especially AtCPK1 and AtCPK2. Northern analysis of steady-state mRNA levels for CpCPK1 in etiolated and light-grown zucchini seedlings shows that the transcript is most abundant in etiolated hypocotyls and overall expression is suppressed by light. As described for other members of the CDPK gene family from different species, the CpCPK1 clone has a putative N-terminal myristoylation sequence. In this study, site-directed mutagenesis and an in vitro coupled transcription/translation system were used to demonstrate that the protein encoded by this cDNA is specifically myristoylated by a plant N-myristoyl transferase. This is the first demonstration of myristoylation of a CDPK protein which may contribute to the mechanism by which this protein is localized to the plasma membrane.

Arsenic trioxide inhibits Ewing's sarcoma cell invasiveness by targeting p38(MAPK) and c-Jun N-terminal kinase.

PubMed

Zhang, Shuai; Guo, Wei; Ren, Ting-Ting; Lu, Xin-Chang; Tang, Guo-Qing; Zhao, Fu-Long

2012-01-01

Ewing's sarcoma is the second most frequent primary malignant bone tumor, mainly affecting children and young adults. The notorious metastatic capability of this tumor aggravates patient mortality and remains a problem to be overcome. We investigated the effect of arsenic trioxide (As₂O₃) on the metastasis capability of Ewing's sarcoma cells. We performed 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide assays to choose appropriate concentrations of As₂O₃ for the experiments. Migration, invasion, and adhesion assays were performed to assess the effect of As₂O₃ on the metastasis of Ewing's sarcoma. Immunofluorescent staining was used to observe cytoskeleton reorganization in Ewing's sarcoma cells treated with As₂O₃. Changes in matrix metalloproteinase-9 expression and the mitogen-activated protein kinase (MAPK) pathway were investigated using western blot. Inhibitors of p38(MAPK) (sb202190) and c-Jun NH₂-terminal kinase (JNK, sp600125) were used in invasion assays to determine the effect of p38(MAPK) and JNK. We found that As₂O₃ may markedly inhibit the migration and invasion capacity of Ewing's sarcoma cells with structural rearrangements of the actin cytoskeleton. The expressions of matrix metalloproteinase-9, phosphor-p38(MAPK), and phosphor-JNK were suppressed by As₂O₃ treatment in a dose-dependent manner. The inhibitors of p38(MAPK) (sb202190) and JNK (sp600125) enhanced the inhibition induced by As₂O₃, which was counteracted by anisomycin, an activating agent of p38(MAPK) and JNK. Taken together, our results demonstrate that As₂O₃ can inhibit the metastasis capability of RD-ES and A-673 cells and may have new therapeutic value for Ewing's sarcoma.