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Sample records for active n-terminal kinase

  1. Activation of c-Jun N-terminal Kinases by Ribotoxic Stresses

    Institute of Scientific and Technical Information of China (English)

    Dong-Yun Ouyang; Yuan-Yuan Wang; Yong-Tang Zheng

    2005-01-01

    The c-Jun N-terminal kinases (JNKs) are classic stress-activated protein kinases. Many cellular stresses have been shown to stimulate JNK activation. In this review, we focus on ribotoxic stresses based on their multiple biological potencies including anti-HIV-1 activity. Some of the functions of ribotoxins and the signaling transduction pathway that mediated are mentioned. Different from other stimulators, ribotoxic stresses act on special motifs of 28S rRNA in translationally active mammal ribosomes. Binding and damaging on the motif leads to JNK activation and subsequently biological response to the signal initiator, which is named ribotoxic stress response.

  2. Activation of G Protein-Coupled Receptor Kinase 1 Involves Interactions between Its N-Terminal Region and Its Kinase Domain

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chih-chin; Orban, Tivadar; Jastrzebska, Beata; Palczewski, Krzysztof; Tesmer, John J.G. (Case Western); (Michigan)

    2012-03-16

    G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs) to initiate receptor desensitization. In addition to the canonical phosphoacceptor site of the kinase domain, activated receptors bind to a distinct docking site that confers higher affinity and activates GRKs allosterically. Recent mutagenesis and structural studies support a model in which receptor docking activates a GRK by stabilizing the interaction of its 20-amino acid N-terminal region with the kinase domain. This interaction in turn stabilizes a closed, more active conformation of the enzyme. To investigate the importance of this interaction for the process of GRK activation, we first validated the functionality of the N-terminal region in rhodopsin kinase (GRK1) by site-directed mutagenesis and then introduced a disulfide bond to cross-link the N-terminal region of GRK1 with its specific binding site on the kinase domain. Characterization of the kinetic and biophysical properties of the cross-linked protein showed that disulfide bond formation greatly enhances the catalytic efficiency of the peptide phosphorylation, but receptor-dependent phosphorylation, Meta II stabilization, and inhibition of transducin activation were unaffected. These data indicate that the interaction of the N-terminal region with the kinase domain is important for GRK activation but does not dictate the affinity of GRKs for activated receptors.

  3. Melanocortin-4 receptor activation inhibits c-Jun N-terminal kinase activity and promotes insulin signaling

    OpenAIRE

    Chai, Biaoxin; Li, Ji-Yao; Zhang, Weizhen; Wang, Hui; Mulholland, Michael W.

    2009-01-01

    The melanocortin system is crucial to regulation of energy homeostasis. The melanocortin receptor type 4 (MC4R) modulates insulin signaling via effects on c-Jun N-terminal kinase (JNK). The melanocortin agonist NDP-MSH dose-dependently inhibited JNK activity in HEK293 cells stably expressing the human MC4R; effects were reversed by melanocortin receptor antagonist. NDP-MSH time- and dose-dependently inhibited IRS-1ser307 phosphorylation, effects also reversed by a specific melanocortin recept...

  4. A novel human STE20-related protein kinase, HGK, that specifically activates the c-Jun N-terminal kinase signaling pathway.

    Science.gov (United States)

    Yao, Z; Zhou, G; Wang, X S; Brown, A; Diener, K; Gan, H; Tan, T H

    1999-01-22

    The yeast serine/threonine kinase STE20 activates a signaling cascade that includes STE11 (mitogen-activated protein kinase kinase kinase), STE7 (mitogen-activated protein kinase kinase), and FUS3/KSS1 (mitogen-activated protein kinase) in response to signals from both Cdc42 and the heterotrimeric G proteins associated with transmembrane pheromone receptors. Using degenerate polymerase chain reaction, we have isolated a human cDNA encoding a protein kinase homologous to STE20. This protein kinase, designated HPK/GCK-like kinase (HGK), has nucleotide sequences that encode an open reading frame of 1165 amino acids with 11 kinase subdomains. HGK was a serine/threonine protein kinase that specifically activated the c-Jun N-terminal kinase (JNK) signaling pathway when transfected into 293T cells, but it did not stimulate either the extracellular signal-regulated kinase or p38 kinase pathway. HGK also increased AP-1-mediated transcriptional activity in vivo. HGK-induced JNK activation was inhibited by the dominant-negative MKK4 and MKK7 mutants. The dominant-negative mutant of TAK1, but not MEKK1 or MAPK upstream kinase (MUK), strongly inhibited HGK-induced JNK activation. TNF-alpha activated HGK in 293T cells, as well as the dominant-negative HGK mutants, inhibited TNF-alpha-induced JNK activation. These results indicate that HGK, a novel activator of the JNK pathway, may function through TAK1, and that the HGK --> TAK1 --> MKK4, MKK7 --> JNK kinase cascade may mediate the TNF-alpha signaling pathway. PMID:9890973

  5. Hippocampal activation of c-Jun N-terminal kinase,protein kinase B,and p38 mitogen-activated protein kinase in a chronic stress rat model of depression

    Institute of Scientific and Technical Information of China (English)

    Wei Dai; Weidong Li; Jun Lu; Yingge A; Ya Tu

    2010-01-01

    Recent studies have shown that vaned stress stimuli activate c-Jun N-terminal kinase(JNK),protein kinase B(Akt),and p38 mitogen-activated protein kinase(p38)signal transduction pathway,and also regulate various apoptotic cascades.JNK and p38 promote apoptosis,but Akt protects against apoptosis,in hippocampal neurons.However,changes in the transduction pathway in different regions of brain tissues in a chronic stress rat model of depression remain poorly understood.Results from this study showed that JNK phosphorylation levels were significantly greater in the stress group hippocampus compared with the control group(P 0.05).These results suggested that the JNK signal pathway is activated by JNK phosphorylation and participates in pathophysiological changes in rat models of depression.

  6. TAp73-mediated the activation of c-Jun N-terminal kinase enhances cellular chemosensitivity to cisplatin in ovarian cancer cells.

    Directory of Open Access Journals (Sweden)

    Pingde Zhang

    Full Text Available P73, one member of the tumor suppressor p53 family, shares highly structural and functional similarity to p53. Like p53, the transcriptionally active TAp73 can mediate cellular response to chemotherapeutic agents in human cancer cells by up-regulating the expressions of its pro-apoptotic target genes such as PUMA, Bax, NOXA. Here, we demonstrated a novel molecular mechanism for TAp73-mediated apoptosis in response to cisplatin in ovarian cancer cells, and that was irrespective of p53 status. We found that TAp73 acted as an activator of the c-Jun N-terminal kinase (JNK signaling pathway by up-regulating the expression of its target growth arrest and DNA-damage-inducible protein GADD45 alpha (GADD45α and subsequently activating mitogen-activated protein kinase kinase-4 (MKK4. Inhibition of JNK activity by a specific inhibitor or small interfering RNA (siRNA significantly abrogated TAp73-mediated apoptosis induced by cisplatin. Furthermore, inhibition of GADD45α by siRNA inactivated MKK4/JNK activities and also blocked TAp73-mediated apoptosis induction by cisplatin. Our study has demonstrated that TAp73 activated the JNK apoptotic signaling pathway in response to cisplatin in ovarian cancer cells.

  7. Cytotoxic Activity of 3,6-Dihydroxyflavone in Human Cervical Cancer Cells and Its Therapeutic Effect on c-Jun N-Terminal Kinase Inhibition

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    Eunjung Lee

    2014-08-01

    Full Text Available Previously we have shown that 3,6-dihydroxyflavone (3,6-DHF is a potent agonist of the human peroxisome proliferator-activated receptor (hPPAR with cytotoxic effects on human cervical cancer cells. To date, the mechanisms by which 3,6-DHF exerts its antitumor effects on cervical cells have not been clearly defined. Here, we demonstrated that 3,6-DHF exhibits a novel antitumor activity against HeLa cells with IC50 values of 25 μM and 9.8 μM after 24 h and 48 h, respectively. We also showed that the anticancer effects of 3,6-DHF are mediated via the toll-like receptor (TLR 4/CD14, p38 mitogen-activated protein kinase (MAPK, Jun-N terminal kinase (JNK, extracellular-signaling regulated kinase (ERK, and cyclooxygenase (COX-2 pathways in lipopolysaccharide (LPS-stimulated RAW264.7 cells. We found that 3,6-DHF showed a similar IC50 (113 nM value to that of the JNK inhibitor, SP600125 (IC50 = 118 nM in a JNK1 kinase assay. Binding studies revealed that 3,6-DHF had a strong binding affinity to JNK1 (1.996 × 105 M−1 and that the 6-OH and the carbonyl oxygen of the C ring of 3,6-DHF participated in hydrogen bonding interactions with the carbonyl oxygen and the amide proton of Met111, respectively. Therefore, 3,6-DHF may be a candidate inhibitor of JNKs, with potent anticancer effects.

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

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    Wang Wen

    2011-01-01

    Full Text Available Abstract 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.

  9. Inhibition of Apoptosis in Prostate Cancer Cells by Androgens Is Mediated through Downregulation of c-Jun N-terminal Kinase Activation

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    Petra Isabel Lorenzo

    2008-05-01

    Full Text Available Androgen deprivation induces the regression of prostate tumors mainly due to an increase in the apoptosis rate; however, the molecular mechanisms underlying the antiapoptotic actions of androgens are not completely understood. We have studied the antiapoptotic effects of androgens in prostate cancer cells exposed to different proapoptotic stimuli. Terminal deoxynucleotidyl transferase-mediated nick-end labeling and nuclear fragmentation analyses demonstrated that androgens protect LNCaP prostate cancer cells from apoptosis induced by thapsigargin, the phorbol ester 12-O-tetradecanoyl-13-phorbol-acetate, or UV irradiation. These three stimuli require the activation of the c-Jun N-terminal kinase (JNK pathway to induce apoptosis and in all three cases, androgen treatment blocks JNK activation. Interestingly, okadaic acid, a phosphatase inhibitor that causes apoptosis in LNCaP cells, induces JNK activation that is also inhibited by androgens. Actinomycin D, the antiandrogen bicalutamide or specific androgen receptor (AR knockdown by small interfering RNA all blocked the inhibition of JNK activation mediated by androgens indicating that this activity requires AR-dependent transcriptional activation. These data suggest that the crosstalk between AR and JNK pathways may have important implications in prostate cancer progression and may provide targets for the development of new therapies.

  10. Induction of apoptosis by casticin in cervical cancer cells: reactive oxygen species-dependent sustained activation of Jun N-terminal kinase

    Institute of Scientific and Technical Information of China (English)

    Fanxiang Zeng; Li Tian; Fei Liu; Jianguo Cao; Meifang Quan; Xifeng Sheng

    2012-01-01

    Casticin,a polymethoxyflavone from Fructus viticis used as an anti-inflammatory agent in Chinese traditional medicine,has been reported to have anti-cancer activity.The purpose of this study was to examine the apoptotic activity of casticin on human cervical cancer cells and its molecular mechanism.We revealed a novel mechanism by which casticin-induced apoptosis occurs and showed for the first time that the apoptosis induced by casticin is mediated through generation of reactive oxygen species (ROS) and sustained activation of c-Jun N-terminal kinase (JNK) in HeLa cells.Casticin markedly increased the levels of intracellular ROS and induced the expression of phosphorylated JNK and cJun protein.Pre-treatment with N-acetylcvsteine and SP600125 effectively attenuated induction of apoptosis by casticin in HeLa cells.Moreover,casticin induced ROS production and apoptotic cell death in other cervical cancer cell lines,such as CasKi and SiHa.Importantly,casticin did not cause generation of ROS or induction of apoptosis in normal human peripheral blood mononuclear cells and embryonic kidney epithelium 293 cells.These results suggest that ROS generation and sustained JNK activation by casticin play a role in casticin-induced apoptosis and raise the possibility that treatment with casticin might be promising as a new therapy against human cervical cancer.

  11. Monosodium Urate in the Presence of RANKL Promotes Osteoclast Formation through Activation of c-Jun N-Terminal Kinase

    Directory of Open Access Journals (Sweden)

    Jung-Yoon Choe

    2015-01-01

    Full Text Available The aim of this study was to clarify the role of monosodium urate (MSU crystals in receptor activator of nuclear factor kB ligand- (RANKL- RANK-induced osteoclast formation. RAW 264.7 murine macrophage cells were incubated with MSU crystals or RANKL and differentiated into osteoclast-like cells as confirmed by staining for tartrate-resistant acid phosphatase (TRAP and actin ring, pit formation assay, and TRAP activity assay. MSU crystals in the presence of RANKL augmented osteoclast differentiation, with enhanced mRNA expression of NFATc1, cathepsin K, carbonic anhydrase II, and matrix metalloproteinase-9 (MMP-9, in comparison to RAW 264.7 macrophages incubated in the presence of RANKL alone. Treatment with both MSU crystals and RANKL induced osteoclast differentiation by activating downstream molecules in the RANKL-RANK pathway including tumor necrosis factor receptor-associated factor 6 (TRAF-6, JNK, c-Jun, and NFATc1. IL-1b produced in response to treatment with both MSU and RANKL is involved in osteoclast differentiation in part through the induction of TRAF-6 downstream of the IL-1b pathway. This study revealed that MSU crystals contribute to enhanced osteoclast formation through activation of RANKL-mediated pathways and recruitment of IL-1b. These findings suggest that MSU crystals might be a pathologic causative agent of bone destruction in gout.

  12. S-adenosyl-methionine decreases ethanol-induced apoptosis in primary hepatocyte cultures by a c-Jun N-terminal kinase activity-independent mechanism

    Institute of Scientific and Technical Information of China (English)

    María del Pilar Cabrales-Romero; Lucrecia Márquez-Rosado; Samia Fattel-Fazenda; Cristina Trejo-Solís; Evelia Arce-Popoca; Leticia Alemén-Lazarini; Saúl Villa-Trevi(n)o

    2006-01-01

    AIM: To determine the role of c-Jun N-terminal kinase (JNK) activity in ethanol-induced apoptosis and the modulation of this signaling cascade by S-Adenosylmethionine (AdoMet).METHODS: Primary hepatocyte cultures were pretreated with 100 μmol/L SP600125, a selective JNK inhibitor, 1 mL/L DMSO or 4 mmol/L AdoMet and then exposed to 100 mmo/L ethanol. Hepatocyte apoptosis was determined by the TUNEL and DNA ladder assays.JNK activity and its inhibition by SP600125 and AdoMet were determined by Western blot analysis of c-jun phosphorylation and Bid fragmentation. SP600125 and AdoMet effects on the apoptotic signaling pathway were determined by Western blot analysis of cytochrome c release and pro-caspase 3 fragmentation. The AdoMet effect on glutathione levels was measured by Ellman's method and reactive oxygen species (ROS) generation by cell cytometry.RESULTS: The exposure of hepatocytes to ethanol induced JNK activation, c-jun phosphorylation, Bid fragmentation, cytochrome c release and pro-caspase 3 cleavage; these effects were diminished by SP600125, and caused a significant decreasein ethanol-induced apoptosis (P< 0.05). AdoMet exerted an antioxidant effect maintaining glutathione levels and decreasing ROS generation, without a significant effect on JNK activity,and prevented cytochrome c release and pro-caspase 3 cleavage.CONCLUSION: The JNK signaling cascade is a key component of the proapoptotic signaling pathway induced by ethanol. JNK activation may be independent from ROS generation, since AdoMet which exerted antioxidant properties did not have a significant effect on JNK activity. JNK pathway modulator agents and AdoMet may be components of promising therapies for alcoholic liver disease (ALD) treatment.

  13. Hydrogen-Rich Saline Attenuates Lipopolysaccharide-Induced Heart Dysfunction by Restoring Fatty Acid Oxidation in Rats by Mitigating C-Jun N-Terminal Kinase Activation.

    Science.gov (United States)

    Tao, Bingdong; Liu, Lidan; Wang, Ni; Tong, Dongyi; Wang, Wei; Zhang, Jin

    2015-12-01

    Sepsis is common in intensive care units (ICU) and is associated with high mortality. Cardiac dysfunction complicating sepsis is one of the most important causes of this mortality. This dysfunction is due to myocardial inflammation and reduced production of energy by the heart. A number of studies have shown that hydrogen-rich saline (HRS) has a beneficial effect on sepsis. Therefore, we tested whether HRS prevents cardiac dysfunction by increasing cardiac energy. Four groups of rats received intraperitoneal injections of one of the following solutions: normal saline (NS), HRS, lipopolysaccharide (LPS), and LPS plus HRS. Cardiac function was measured by echocardiography 8 h after the injections. Gene and protein expression related to fatty acid oxidation (FAO) were measured by quantitative polymerase chain reaction (PCR) and Western blot analysis. The injection of LPS compromised heart function through decreased fractional shortening (FS) and increased left ventricular diameter (LVD). The addition of HRS increased FS, palmitate triphosphate, and the ratio of phosphocreatinine (PCr) to adenosine triphosphate (ATP) as well as decreasing LVD. The LPS challenge reduced the expression of genes related to FAO, including perioxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), perioxisome proliferator-activated receptor alpha (PPARα), Estrogen-related receptor alpha (ERRα), and their downstream targets, in mRNA and protein level, which were attenuated by HRS. However, HRS had little effect on glucose metabolism. Furthermore, HRS inhibited c-Jun N-terminal kinase (JNK) activation in the rat heart. Inhibition of JNK by HRS showed beneficial effects on LPS-challenged rats, at least in part, by restoring cardiac FAO.

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

    Energy Technology Data Exchange (ETDEWEB)

    Du, Kuo; Williams, C. David; McGill, Mitchell R.; Xie, Yuchao [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Farhood, Anwar [Department of Pathology, St. David' s North Austin Medical Center, Austin, TX 78756 (United States); Vinken, Mathieu [Department of Toxicology, Center for Pharmaceutical Sciences, Vrije Universiteit Brussels, 1090 Brussels (Belgium); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

    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 when 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-APB

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

    International Nuclear Information System (INIS)

    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 when 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-APB

  16. Correlation between spina bifida manifesta in fetal rats and c-Jun N-terminal kinase signaling

    Institute of Scientific and Technical Information of China (English)

    Yinghuan Ma; Yongxin Bao; Chenghao Li; Fubin Jiao; Hongjie Xin; Zhengwei Yuan

    2012-01-01

    Fetal rat models with neural tube defects were established by injection with retinoic acid at 10 days after conception. The immunofluorescence assay and western blot analysis showed that the number of caspase-3 positive cells in myeloid tissues for spina bifida manifesta was increased. There was also increased phosphorylation of c-Jun N-terminal kinase, a member of the mitogen activated protein kinase family. The c-Jun N-terminal kinase phosphorylation level was positively correlated with caspase-3 expression in myeloid tissues for spina bifida manifesta. Experimental findings indicate that abnormal apoptosis is involved in retinoic acid-induced dominant spina bifida formation in fetal rats, and may be associated with the c-Jun N-terminal kinase signal transduction pathway.

  17. Tumor necrosis factor alpha promotes the proliferation of human nucleus pulposus cells via nuclear factor-κB, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase.

    Science.gov (United States)

    Wang, Xiao-Hu; Hong, Xin; Zhu, Lei; Wang, Yun-Tao; Bao, Jun-Ping; Liu, Lei; Wang, Feng; Wu, Xiao-Tao

    2015-04-01

    Although tumor necrosis factor alpha (TNF-α) is known to play a critical role in intervertebral disc (IVD) degeneration, the effect of TNF-α on nucleus pulposus (NP) cells has not yet been elucidated. The aim of this study was to explore the effect of TNF-α on proliferation of human NP cells. NP cells were treated with different concentrations of TNF-α. Cell proliferation was determined by cell counting kit-8 (CCK-8) analysis and Ki67 immunofluorescence staining, and expression of cyclin B1 was studied by quantitative real-time RT-PCR. Cell cycle was measured by flow cytometry and cell apoptosis was analyzed using an Annexin V-fluorescein isothiocyanate (FITC) & propidium iodide (PI) apoptosis detection kit. To identify the mechanism by which TNF-α induced proliferation of NP cells, selective inhibitors of major signaling pathways were used and Western blotting was carried out. Treatment with TNF-α increased cell viability (as determined by CCK-8 analysis) and expression of cyclin B1 and the number of Ki67-positive and S-phase NP cells, indicating enhancement of proliferation. Consistent with this, NP cell apoptosis was suppressed by TNF-α treatment. Moreover, inhibition of NF-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) blocked TNF-α-stimulated proliferation of NP cells. In conclusion, the current findings suggest that the effect of TNF-α on IVD degeneration involves promotion of the proliferation of human NP cells via the NF-κB, JNK, and p38 MAPK pathways.

  18. Regulation of Apoptotic c-Jun N-Terminal Kinase Signaling by a Stabilization-Based Feed-Forward Loop†

    OpenAIRE

    Xu, Zhiheng; Kukekov, Nikolay V.; Greene, Lloyd A.

    2005-01-01

    A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect ...

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

    OpenAIRE

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

    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 le...

  20. Free cholesterol-induced macrophage apoptosis is mediated by inositol-requiring enzyme 1 alpha-regulated activation of Jun N-terminal kinase

    Institute of Scientific and Technical Information of China (English)

    Fangming Li; Yi Guo; Shenggang Sun; Xin Jiang; Bingshan Tang; Qizhang Wang; Ling Wang

    2008-01-01

    Macrophage death in advanced atherosclerotic lesions leads to iesional necrosis, possible plaque rupture, and acute vascular occlusion. A likely cause of macrophage death is the accumulation of free cholesterol (FC) leading to activation of endoplasmic reticulum (ER) stress-induced apoptosis.Inositol-requiring enzyme 1 alpha (IRE1α) is an integral membrane protein of the ER that is a key signaling step in cholesterol-induced apoptosis in macrophages, activated by stress in the ER. However, the role of IRE1α in the regulation of ER stress-induced macrophage death and the mechanism for this process are largely unclear.In this study,a cell culture model was used to explore the mechanisms involved in the ER stress pathway of FC-induced macrophage death.The results herein showed that FC loading of macrophages leads to an apoptotic response that is partially dependent on initiation by activation of IRE1α.Taken together,these results showed that the IRE1-apoptosis-signaling kinase 1-c-Jun NH2-terminal kinase cascade pathway was required in this process.Moreover,the data suggested a novel cellular mechanism for cholesterol-induced macrophage death in advanced atherosclerotic lesions.The critical function of this signaling cascade is indicated by prevention of ER stress-induced apoptosis after inhibition of IRE1α,or c-Jun NH2-terminal kinase.

  1. Highly Oxygenated Sesquiterpene Lactones from Cousinia aitchisonii and their Cytotoxic Properties: Rhaserolide Induces Apoptosis in Human T Lymphocyte (Jurkat) Cells via the Activation of c-Jun n-terminal Kinase Phosphorylation.

    Science.gov (United States)

    Iranshahy, Milad; Tayarani-Najaran, Zahra; Kasaian, Jamal; Ghandadi, Morteza; Emami, Seyed Ahmad; Asili, Javad; Chandran, Jima N; Schneider, Bernd; Iranshahi, Mehrdad

    2016-02-01

    Infrared-guided chromatographic fractionation of sesquiterpene lactones from the extracts of Cousinia aitchisonii and Cousinia concolor led to the isolation of five pure compounds. A new sesquiterpene lactone, namely, aitchisonolide, and two known sesquiterpene lactones (desoxyjanerin and rhaserolide) were isolated from C. aitchisonii and two known lignans (arctiin and arctigenin) from C. concolor. The structures of these compounds were elucidated by one-dimensional and two-dimensional nuclear magnetic resonance techniques, as well as high-resolution mass spectrometry. The purified and characterized compounds were subjected to cytotoxicity assay. The sesquiterpene lactones desoxyjanerin and rhaserolide showed significant cytotoxic activities against five different cancer cell lines and the normal human embryonic kidney cell line. Rhaserolide was chosen to evaluate the possible mechanism of action. Western blot analysis revealed that rhaserolide could induce apoptosis in Jurkat cells via the activation of c-Jun n-terminal kinase phosphorylation. PMID:26581585

  2. Highly Oxygenated Sesquiterpene Lactones from Cousinia aitchisonii and their Cytotoxic Properties: Rhaserolide Induces Apoptosis in Human T Lymphocyte (Jurkat) Cells via the Activation of c-Jun n-terminal Kinase Phosphorylation.

    Science.gov (United States)

    Iranshahy, Milad; Tayarani-Najaran, Zahra; Kasaian, Jamal; Ghandadi, Morteza; Emami, Seyed Ahmad; Asili, Javad; Chandran, Jima N; Schneider, Bernd; Iranshahi, Mehrdad

    2016-02-01

    Infrared-guided chromatographic fractionation of sesquiterpene lactones from the extracts of Cousinia aitchisonii and Cousinia concolor led to the isolation of five pure compounds. A new sesquiterpene lactone, namely, aitchisonolide, and two known sesquiterpene lactones (desoxyjanerin and rhaserolide) were isolated from C. aitchisonii and two known lignans (arctiin and arctigenin) from C. concolor. The structures of these compounds were elucidated by one-dimensional and two-dimensional nuclear magnetic resonance techniques, as well as high-resolution mass spectrometry. The purified and characterized compounds were subjected to cytotoxicity assay. The sesquiterpene lactones desoxyjanerin and rhaserolide showed significant cytotoxic activities against five different cancer cell lines and the normal human embryonic kidney cell line. Rhaserolide was chosen to evaluate the possible mechanism of action. Western blot analysis revealed that rhaserolide could induce apoptosis in Jurkat cells via the activation of c-Jun n-terminal kinase phosphorylation.

  3. Calcium has a permissive role in interleukin-1beta-induced c-jun N-terminal kinase activation in insulin-secreting cells

    DEFF Research Database (Denmark)

    Størling, Joachim; Zaitsev, Sergei V; Kapelioukh, Iouri L;

    2005-01-01

    -acetoxymethyl], an inhibitor of calmodulin (W7), and inhibitors of Ca(2+)/calmodulin-dependent kinase (KN62 and KN93) partially reduced IL-1beta-stimulated c-jun phosphorylation in INS-1 or betaTC3 cells. Our data suggest that Ca(2+) plays a permissive role in IL-1beta activation of the JNK signaling pathway in insulin......-cells are largely unknown. In this study, we investigated whether Ca(2+) plays a role for IL-1beta-induced JNK activation. In insulin-secreting rat INS-1 cells cultured in the presence of 11 mm glucose, combined pharmacological blockade of L- and T-type Ca(2+) channels suppressed IL-1beta-induced in vitro...

  4. c-Jun N-terminal Kinase (JNK) signaling as a therapeutic target for Alzheimer’s disease

    OpenAIRE

    Yarza, Ramon; Vela, Silvia; Solas, Maite; Ramirez, Maria J

    2016-01-01

    c-Jun N-terminal kinases (JNKs) are a family of protein kinases that play a central role in stress signaling pathways implicated in gene expression, neuronal plasticity, regeneration, cell death, and regulation of cellular senescence. It has been shown that there is a JNK pathway activation after exposure to different stressing factors, including cytokines, growth factors, oxidative stress, unfolded protein response signals or Aβ peptides. Altogether, JNKs have become a focus of screening str...

  5. Cadmium induces apoptosis in pancreatic β-cells through a mitochondria-dependent pathway: the role of oxidative stress-mediated c-Jun N-terminal kinase activation.

    Directory of Open Access Journals (Sweden)

    Kai-Chih Chang

    Full Text Available Cadmium (Cd, one of well-known highly toxic environmental and industrial pollutants, causes a number of adverse health effects and diseases in humans. The growing epidemiological studies have suggested a possible link between Cd exposure and diabetes mellitus (DM. However, the toxicological effects and underlying mechanisms of Cd-induced pancreatic β-cell injury are still unknown. In this study, we found that Cd significantly decreased cell viability, and increased sub-G1 hypodiploid cells and annexin V-Cy3 binding in pancreatic β-cell-derived RIN-m5F cells. Cd also increased intracellular reactive oxygen species (ROS generation and malondialdehyde (MDA production and induced mitochondrial dysfunction (the loss of mitochondrial membrane potential (MMP and the increase of cytosolic cytochrome c release, the decreased Bcl-2 expression, increased p53 expression, poly (ADP-ribose polymerase (PARP cleavage, and caspase cascades, which accompanied with intracellular Cd accumulation. Pretreatment with the antioxidant N-acetylcysteine (NAC effectively reversed these Cd-induced events. Furthermore, exposure to Cd induced the phosphorylations of c-jun N-terminal kinases (JNK, extracellular signal-regulated kinases (ERK1/2, and p38-mitogen-activated protein kinase (MAPK, which was prevented by NAC. Additionally, the specific JNK inhibitor SP600125 or JNK-specific small interference RNA (si-RNA transfection suppressed Cd-induced β-cell apoptosis and related signals, but not ERK1/2 and p38-MAPK inhibitors (PD98059 and SB203580 did not. However, the JNK inhibitor or JNK-specific si-RNA did not suppress ROS generation in Cd-treated cells. These results indicate that Cd induces pancreatic β-cell death via an oxidative stress downstream-mediated JNK activation-triggered mitochondria-regulated apoptotic pathway.

  6. Jun N-Terminal Protein Kinase Enhances Middle Ear Mucosal Proliferation during Bacterial Otitis Media▿

    Science.gov (United States)

    Furukawa, Masayuki; Ebmeyer, Jörg; Pak, Kwang; Austin, Darrell A.; Melhus, Åsa; Webster, Nicholas J. G.; Ryan, Allen F.

    2007-01-01

    Mucosal hyperplasia is a characteristic component of otitis media. The present study investigated the participation of signaling via the Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase in middle ear mucosal hyperplasia in animal models of bacterial otitis media. Otitis media was induced by the inoculation of nontypeable Haemophilus influenzae into the middle ear cavity. Western blotting revealed that phosphorylation of JNK isoforms in the middle ear mucosa preceded but paralleled mucosal hyperplasia in this in vivo rat model. Nuclear JNK phosphorylation was observed in many cells of both the mucosal epithelium and stroma by immunohistochemistry. In an in vitro model of primary rat middle ear mucosal explants, bacterially induced mucosal growth was blocked by the Rac/Cdc42 inhibitor Clostridium difficile toxin B, the mixed-lineage kinase inhibitor CEP11004, and the JNK inhibitor SP600125. Finally, the JNK inhibitor SP600125 significantly inhibited mucosal hyperplasia during in vivo bacterial otitis media in guinea pigs. Inhibition of JNK in vivo resulted in a diminished proliferative response, as shown by a local decrease in proliferating cell nuclear antigen protein expression by immunohistochemistry. We conclude that activation of JNK is a critical pathway for bacterially induced mucosal hyperplasia during otitis media, influencing tissue proliferation. PMID:17325051

  7. c-Jun N-terminal kinase mediates constitutive human eosinophil apoptosis

    OpenAIRE

    Hasala, Hannele; Zhang, Xianzhi; Saarelainen, Seppo; Moilanen, Eeva; Kankaanranta, Hannu

    2007-01-01

    c-Jun N-terminal kinase mediates constitutive human eosinophil apoptosis correspondence: Corresponding author. Tel.: +358335517318; fax: +358335518082. (Kankaanranta, Hannu) (Kankaanranta, Hannu) The Immunopharmacology Research Group--> , Medical School--> , University of Tampere--> , Tampere--> - FINLAND (Hasala, Hannele) The Immunopharmacology Research Group--> , Medical School--...

  8. Activation of Tax protein by c-Jun-N-terminal kinase is not dependent on the presence or absence of the early growth response-1 gene product.

    Science.gov (United States)

    Parra, Eduardo; Gutierréz, Luís; Ferreira, Jorge

    2016-02-01

    The Tax protein of human T cell leukemia virus type 1 plays a major role in the pathogenesis of adult T cell leukemia (ATL), an aggressive neoplasia of CD4+ T cells. In the present study, we investigated whether the EGR-1 pathway is involved in the regulation of Tax-induced JNK expression in human Jurkat T cells transfected to express the Tax protein in the presence or absence of PMA or ionomycin. Overexpression of EGR-1 in Jurkat cells transfected to express Tax, promoted the activation of several genes, with the most potent being those that contained AP-1 (Jun/c-Fos), whereas knockdown of endogenous EGR-1 by small interfering RNA (siRNA) somewhat reduced Tax-mediated JNK-1 transcription. Additionally, luciferase-based AP-1 and NF-κB reporter gene assays demonstrated that inhibition of EGR-1 expression by an siRNA did not affect the transcriptional activity of a consensus sequence of either AP-1 or NF-κB. On the other hand, the apoptosis assay, using all-trans retinoic acid (ATRA) as an inducer of apoptosis, confirmed that siRNA against EGR-1 failed to suppress ATRA-induced apoptosis in Jurkat and Jurkat-Tax cells, as noted by the low levels of both DEVDase activity and DNA fragmentation, indicating that the induction of apoptosis by ATRA was Egr-1-independent. Finally, our data showed that activation of Tax by JNK-1 was not dependent on the EGR-1 cascade of events, suggesting that EGR-1 is important but not a determinant for the activity for Tax-induced proliferation of Jurkat cells.

  9. Activation of Tax protein by c-Jun-N-terminal kinase is not dependent on the presence or absence of the early growth response-1 gene product.

    Science.gov (United States)

    Parra, Eduardo; Gutierréz, Luís; Ferreira, Jorge

    2016-02-01

    The Tax protein of human T cell leukemia virus type 1 plays a major role in the pathogenesis of adult T cell leukemia (ATL), an aggressive neoplasia of CD4+ T cells. In the present study, we investigated whether the EGR-1 pathway is involved in the regulation of Tax-induced JNK expression in human Jurkat T cells transfected to express the Tax protein in the presence or absence of PMA or ionomycin. Overexpression of EGR-1 in Jurkat cells transfected to express Tax, promoted the activation of several genes, with the most potent being those that contained AP-1 (Jun/c-Fos), whereas knockdown of endogenous EGR-1 by small interfering RNA (siRNA) somewhat reduced Tax-mediated JNK-1 transcription. Additionally, luciferase-based AP-1 and NF-κB reporter gene assays demonstrated that inhibition of EGR-1 expression by an siRNA did not affect the transcriptional activity of a consensus sequence of either AP-1 or NF-κB. On the other hand, the apoptosis assay, using all-trans retinoic acid (ATRA) as an inducer of apoptosis, confirmed that siRNA against EGR-1 failed to suppress ATRA-induced apoptosis in Jurkat and Jurkat-Tax cells, as noted by the low levels of both DEVDase activity and DNA fragmentation, indicating that the induction of apoptosis by ATRA was Egr-1-independent. Finally, our data showed that activation of Tax by JNK-1 was not dependent on the EGR-1 cascade of events, suggesting that EGR-1 is important but not a determinant for the activity for Tax-induced proliferation of Jurkat cells. PMID:26573109

  10. 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.

    Directory of Open Access Journals (Sweden)

    Jee-Youn Kim

    Full Text Available 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.

  11. TAp73-mediated the activation of C-jun N-terminal kinase enhances cellular chemosensitivity to cisplatin in ovarian cancer cells

    OpenAIRE

    Pingde Zhang; Stephanie Si Liu; Hextan Yuen Sheung Ngan

    2012-01-01

    P73, one member of the tumor suppressor p53 family, shares highly structural and functional similarity to p53. Like p53, the transcriptionally active TAp73 can mediate cellular response to chemotherapeutic agents in human cancer cells by up-regulating the expressions of its pro-apoptotic target genes such as PUMA, Bax, NOXA. Here, we demonstrated a novel molecular mechanism for TAp73-mediated apoptosis in response to cisplatin in ovarian cancer cells, and that was irrespective of p53 status. ...

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

    Directory of Open Access Journals (Sweden)

    Chang Alice YW

    2012-11-01

    Full Text Available Abstract 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

  13. HEPATIC APOPTOSIS POST-BURN IS MEDIATED BY C-JUN N-TERMINAL KINASE-2

    OpenAIRE

    Marshall, Alexandra H; Brooks, Natasha C; Hiyama, Yaeko; Qa’aty, Nour; Al-mousawi, Ahmed; Finnerty, Celeste C.; Jeschke, Marc G.

    2013-01-01

    The trauma of a severe burn injury induces a hypermetabolic response that increases morbidity and mortality. Previously, our group showed that insulin resistance post-burn injury is associated with endoplasmic reticulum (ER) stress. Evidence suggests that c-jun N-terminal kinase (JNK) -2 may be involved in ER stress-induced apoptosis. Here, we hypothesized that JNK2 contributes to the apoptotic response after burn injury downstream of ER stress. To test this, we compared JNK2 knockout mice (−...

  14. The N-terminal domain of the tomato immune protein Prf contains multiple homotypic and Pto kinase interaction sites.

    Science.gov (United States)

    Saur, Isabel Marie-Luise; Conlan, Brendon Francis; Rathjen, John Paul

    2015-05-01

    Resistance to Pseudomonas syringae bacteria in tomato (Solanum lycopersicum) is conferred by the Prf recognition complex, composed of the nucleotide-binding leucine-rich repeats protein Prf and the protein kinase Pto. The complex is activated by recognition of the P. syringae effectors AvrPto and AvrPtoB. The N-terminal domain is responsible for Prf homodimerization, which brings two Pto kinases into close proximity and holds them in inactive conformation in the absence of either effector. Negative regulation is lost by effector binding to the catalytic cleft of Pto, leading to disruption of its P+1 loop within the activation segment. This change is translated through Prf to a second Pto molecule in the complex. Here we describe a schematic model of the unique Prf N-terminal domain dimer and its interaction with the effector binding determinant Pto. Using heterologous expression in Nicotiana benthamiana, we define multiple sites of N domain homotypic interaction and infer that it forms a parallel dimer folded centrally to enable contact between the N and C termini. Furthermore, we found independent binding sites for Pto at either end of the N-terminal domain. Using the constitutively active mutant ptoL205D, we identify a potential repression site for Pto in the first ∼100 amino acids of Prf. Finally, we find that the Prf leucine-rich repeats domain also binds the N-terminal region, highlighting a possible mechanism for transfer of the effector binding signal to the NB-LRR regulatory unit (consisting of a central nucleotide binding and C-terminal leucine-rich repeats).

  15. Regulation of apoptotic c-Jun N-terminal kinase signaling by a stabilization-based feed-forward loop.

    Science.gov (United States)

    Xu, Zhiheng; Kukekov, Nikolay V; Greene, Lloyd A

    2005-11-01

    A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect occurs through protein stabilization and requires the presence of POSH as well as activation of MLKs and JNKs. Our findings suggest a self-amplifying, feed-forward loop mechanism by which apoptotic stimuli promote the stabilization of JNK pathway components, thereby contributing to cell death. PMID:16260609

  16. Regulation of Apoptotic c-Jun N-Terminal Kinase Signaling by a Stabilization-Based Feed-Forward Loop†

    Science.gov (United States)

    Xu, Zhiheng; Kukekov, Nikolay V.; Greene, Lloyd A.

    2005-01-01

    A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect occurs through protein stabilization and requires the presence of POSH as well as activation of MLKs and JNKs. Our findings suggest a self-amplifying, feed-forward loop mechanism by which apoptotic stimuli promote the stabilization of JNK pathway components, thereby contributing to cell death. PMID:16260609

  17. c-Jun N-terminal kinase phosphorylates DCP1a to control formation of P bodies

    OpenAIRE

    Rzeczkowski, Katharina; Beuerlein, Knut; Müller, Helmut; Dittrich-Breiholz, Oliver; Schneider, Heike; Kettner-Buhrow, Daniela; Holtmann, Helmut; Kracht, Michael

    2011-01-01

    Cytokines and stress-inducing stimuli signal through c-Jun N-terminal kinase (JNK) using a diverse and only partially defined set of downstream effectors. In this paper, the decapping complex subunit DCP1a was identified as a novel JNK target. JNK phosphorylated DCP1a at residue S315 in vivo and in vitro and coimmunoprecipitated and colocalized with DCP1a in processing bodies (P bodies). Sustained JNK activation by several different inducers led to DCP1a dispersion from P bodies, whereas IL-1...

  18. MORINGA TEA BLOCKS ACUTE LUNG INFLAMMATION INDUCED BY SWINE CONFINEMENT DUST THROUGH A MECHANISM INVOLVING TNF-α EXPRESSION, C-JUN N-TERMINAL KINASE ACTIVATION AND NEUTROPHIL REGULATION

    Directory of Open Access Journals (Sweden)

    Mykea Mcknight

    2014-01-01

    Full Text Available Plant based products represent a promising alternative to conventional treatments for inflammation. Moringa oleifera Lam is a tree rich in proteins, vitamins, minerals and a variety of phytochemcals with health benefits. Among the reported health benefits are antioxidant and anti-inflammatory properties. The purpose of this study was to investigate whether tea brewed from dried Moringa leaves would abrogate inflammation in a mouse model of acute lung inflammation induced by LPS or extracts prepared from dust collected from a swine confinement facility (DE. Mice were offered water or Moringa tea for seven days. Tea consumption was significantly greater than that of water consumption on days 1 and 6, but there were no significant differences in weight gain or food consumption. On day seven, mice from both groups were forced to inhale, via intranasal challenge, either Phosphate Buffered Saline (PBS, Lipopolysaccharide (LPS [10 µg mL-1] or DE [10%]. Compared to mice that drank water, mice that drank Moringa tea had significantly less protein (p<0.05 and cellular influx (p<0.0001 into the lung after inhalation of 10% DE. No difference in neutrophil migration into the lungs of water and M. tea groups after LPS or DE challenge was detected. But mice that drank tea had significantly (p<0.05 more neutrophils with apoptotic morphology after DE challenge. TNF-α expression 24 h after inhalation of 10% DE, was significantly higher (p<0.05 in lungs of M. tea mouse group as compared to water group. This increase in TNF-α was accompanied by higher levels of pro and anti-inflammatory cytokines. Finally, activation of c-Jun N-terminal Kinase (JNK in lungs of M. tea+DE group 24 h post inhalation was decreased. Taken together these results suggest that Moringa oleifera leaf tea exerts anti-inflammatory properties on acute lung inflammation induced by swine confinement dust through a mechanism involving neutrophil regulation and JNK

  19. HEPATIC APOPTOSIS POST-BURN IS MEDIATED BY C-JUN N-TERMINAL KINASE-2

    Science.gov (United States)

    Marshall, Alexandra H.; Brooks, Natasha C.; Hiyama, Yaeko; Qa’aty, Nour; Al-mousawi, Ahmed; Finnerty, Celeste C.; Jeschke, Marc G.

    2013-01-01

    The trauma of a severe burn injury induces a hypermetabolic response that increases morbidity and mortality. Previously, our group showed that insulin resistance post-burn injury is associated with endoplasmic reticulum (ER) stress. Evidence suggests that c-jun N-terminal kinase (JNK) -2 may be involved in ER stress-induced apoptosis. Here, we hypothesized that JNK2 contributes to the apoptotic response after burn injury downstream of ER stress. To test this, we compared JNK2 knockout mice (−/−) to wildtype mice after inducing a 30% total body surface area thermal injury. Animals were sacrificed after 1, 3 and 5 days. Inflammatory cytokines in the blood were measured by multiplex analysis. Hepatic ER stress and insulin signaling were assessed by Western Blotting and insulin resistance was measured by a peritoneal glucose tolerance test. Apoptosis in the liver was quantified by TUNEL staining. Liver function was quantified by AST and ALT activity assays. ER stress increased after burn in both JNK2−/− and wildtype mice, indicating that JNK2 activation is downstream of ER stress. Knockout of JNK2 did not affect serum inflammatory cytokines; however, the increase in IL-6 mRNA expression was prevented in the knockouts. Serum insulin did not significantly increase in the JNK2−/− group. On the other hand, insulin signaling (PI3K/Akt pathway) and glucose tolerance tests did not improve in JNK2−/−. As expected, apoptosis in the liver increased after burn injury in wildtype mice but not in JNK2−/−. AST/ALT activity revealed that liver function recovered more quickly in JNK2−/−. This study indicates that JNK2 is a central mediator of hepatic apoptosis after a severe burn. PMID:23324888

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

    International Nuclear Information System (INIS)

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

  1. Identification of a c-Jun N-terminal kinase-2-dependent signal amplification cascade that regulates c-Myc levels in ras transformation

    DEFF Research Database (Denmark)

    Mathiasen, D.P.; Egebjerg, C.; Andersen, S.H.;

    2012-01-01

    Ras is one of the most frequently activated oncogenes in cancer. Two mitogen-activated protein kinases (MAPKs) are important for ras transformation: extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase 2 (JNK2). Here we present a downstream signal amplification cascade that is...... essential for ras transformation. Previous studies show that ERK-mediated serine 62 phosphorylation protects c-Myc from proteasomal degradation. ERK is, however, not alone sufficient to stabilize c-Myc but requires the cooperation of cancerous inhibitor of protein phosphatase 2A (CIP2A), an oncogene that...

  2. Glycogen synthase kinase 3β sustains invasion of glioblastoma via the focal adhesion kinase, Rac1, and c-Jun N-terminal kinase-mediated pathway.

    Science.gov (United States)

    Chikano, Yuri; Domoto, Takahiro; Furuta, Takuya; Sabit, Hemragul; Kitano-Tamura, Ayako; Pyko, Ilya V; Takino, Takahisa; Sai, Yoshimichi; Hayashi, Yutaka; Sato, Hiroshi; Miyamoto, Ken-ichi; Nakada, Mitsutoshi; Minamoto, Toshinari

    2015-02-01

    The failure of current treatment options for glioblastoma stems from their inability to control tumor cell proliferation and invasion. Biologically targeted therapies offer great hope and one promising target is glycogen synthase kinase-3β (GSK3β), implicated in various diseases, including cancer. We previously reported that inhibition of GSK3β compromises the survival and proliferation of glioblastoma cells, induces their apoptosis, and sensitizes them to temozolomide and radiation. Here, we explore whether GSK3β also contributes to the highly invasive nature of glioblastoma. The effects of GSK3β inhibition on migration and invasion of glioblastoma cells were examined by wound-healing and Transwell assays, as well as in a mouse model of glioblastoma. We also investigated changes in cellular microarchitectures, cytoskeletal components, and proteins responsible for cell motility and invasion. Inhibition of GSK3β attenuated the migration and invasion of glioblastoma cells in vitro and that of tumor cells in a mouse model of glioblastoma. These effects were associated with suppression of the molecular axis involving focal adhesion kinase, guanine nucleotide exchange factors/Rac1 and c-Jun N-terminal kinase. Changes in cellular phenotypes responsible for cell motility and invasion were also observed, including decreased formation of lamellipodia and invadopodium-like microstructures and alterations in the subcellular localization, and activity of Rac1 and F-actin. These changes coincided with decreased expression of matrix metalloproteinases. Our results confirm the potential of GSK3β as an attractive therapeutic target against glioblastoma invasion, thus highlighting a second role in this tumor type in addition to its involvement in chemo- and radioresistance. PMID:25504636

  3. c-Jun-N-terminal kinase 1 is necessary for nicotine-induced enhancement of contextual fear conditioning.

    Science.gov (United States)

    Leach, Prescott T; Kenney, Justin W; Gould, Thomas J

    2016-08-01

    Acute nicotine enhances hippocampus-dependent learning. Identifying how acute nicotine improves learning will aid in understanding how nicotine facilitates the development of maladaptive memories that contribute to drug-seeking behaviors, help development of medications to treat disorders associated with cognitive decline, and advance understanding of the neurobiology of learning and memory. The effects of nicotine on learning may involve recruitment of signaling through the c-Jun N-terminal kinase family (JNK 1-3). Learning in the presence of acute nicotine increases the transcription of mitogen-activated protein kinase 8 (MAPK8, also known as JNK1), likely through a CREB-dependent mechanism. The functional significance of JNK1 in the effects of acute nicotine on learning, however, is unknown. The current studies undertook a backward genetic approach to determine the functional contribution JNK1 protein makes to nicotine-enhanced contextual fear conditioning. JNK1 wildtype (WT) and knockout (KO) mice were administered acute nicotine prior to contextual and cued fear conditioning. 24h later, mice were evaluated for hippocampus-dependent (contextual fear conditioning) and hippocampus-independent (cued fear conditioning) memory. Nicotine selectively enhanced contextual conditioning in WT mice, but not in KO mice. Nicotine had no effect on hippocampus-independent learning in either genotype. JNK1 KO and WT mice given saline showed similar levels of learning. These data suggest that JNK1 may be recruited by nicotine and is functionally necessary for the acute effects of nicotine on learning and memory. PMID:27235579

  4. C-jun N-terminal Kinase-mediated Signaling Is Essential for Staphylococcus Aureus-induced U937 Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Jia-he Wang; Bo Yu; Hui-yan Niu; Hui Li; Yi Zhang; Xin Wang; Ping He

    2009-01-01

    Objective To investigate the effect of SP600125, a specific c-jun N-terminal protein kinase (JNK) inhibitor, on Staphylococcus aureus (S. aureus)-induced U937 cell death and the underlying mechanism. Methods The human monocytic U937 cells were treated with S. aureus at different time with or without SP600125. Cell apoptosis was analyzed by flow cytometry. JNK, Bax, and caspase-3 activities were detected by Western blotting. Results S. aureus induced apoptosis in cultured U937 cells in a time-dependent manner. Expression of Bax and phospho-JNK significantly increased in S. aureus-treated U937 cells, and the level of activated caspase-3 also increased in a time-dependent manner. Inhibition of JNK with SP600125 significantly inhibited S. aureus-induced apoptosis in U937 cells. Conclusions S. aureus can induce apoptosis in U937 cells by phosphorylation of JNK and activation of Bax and caspase-3. SP600125 protects U937 cells from apoptosis induced by S. aureus via inhibiting the activity of JNK.

  5. Molecular clone and characterization of c-Jun N-terminal kinases 2 from orange-spotted grouper, Epinephelus coioides.

    Science.gov (United States)

    Guo, Minglan; Wei, Jingguang; Zhou, Yongcan; Qin, Qiwei

    2016-02-01

    c-Jun N-terminal kinase 2 (JNK2) is a multifunctional mitogen-activated protein kinases involving in cell differentiation and proliferation, apoptosis, immune response and inflammatory conditions. In this study, we reported a new JNK2 (Ec-JNK2) derived from orange-spotted grouper, Epinephelus coioides. The full-length cDNA of Ec-JNK2 was 1920 bp in size, containing a 174 bp 5'-untranslated region (UTR), 483 bp 3'-UTR, and a 1263 bp open reading frame (ORF), which encoded a putative protein of 420 amino acids. The deduced protein sequence of Ec-JNK2 contained a conserved Thr-Pro-Tyr (TPY) motif in the domain of serine/threonine protein kinase (S-TKc). Ec-JNK2 has been found to involve in the immune response to pathogen challenges in vivo, and the infection of Singapore grouper iridovirus (SGIV) in vitro. Immunofluorescence staining showed that Ec-JNK2 was localized in the cytoplasm of grouper spleen (GS) cells, and moved to the nucleus after infecting with SGIV. Ec-JNK2 distributed in all immune-related tissues examined. After challenging with lipopolysaccharide (LPS), SGIV and polyriboinosinic polyribocytidylic acid (poly I:C), the mRNA expression of Ec-JNK2 was significantly (P orange-spotted grouper. Over-expressing Ec-JNK2 in fathead minnow (FHM) cells increased the SGIV infection and replication, while over-expressing the dominant-negative Ec-JNK2Δ181-183 mutant decreased it. These results indicated that Ec-JNK2 could be an important molecule in the successful infection and evasion of SGIV.

  6. Growth arrest- and DNA-damage-inducible 45beta gene inhibits c-Jun N-terminal kinase and extracellular signal-regulated kinase and decreases IL-1beta-induced apoptosis in insulin-producing INS-1E cells

    DEFF Research Database (Denmark)

    Larsen, Claus Morten; Døssing, M G; Papa, S;

    2006-01-01

    IL-1beta is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1beta activates beta cell c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, all of which are members of the mitogen......-activated protein kinase (MAPK) family. Inhibition of JNK prevents IL-1beta-mediated beta cell destruction. In mouse embryo fibroblasts and 3DO T cells, overexpression of the gene encoding growth arrest and DNA-damage-inducible 45beta (Gadd45b) downregulates pro-apoptotic JNK signalling. The aim of this study...

  7. The c-Jun N-terminal Kinase 2 Plays a Dominant Role in Human Epidermal Neoplasia

    Science.gov (United States)

    Ke, Hengning; Harris, Rebecca; Coloff, Jonathan; Jin, Jane Y.; Leshin, Benjamin; de Marval, Paula Miliani; Tao, Shiying; Rathmell, Jeffrey C; Hall, Russell P.; Zhang, Jennifer Y.

    2010-01-01

    The c-Jun N-terminal Kinase (JNK) signaling cascade has been implicated in a wide range of diseases, including cancer. It is unclear how different JNK proteins contribute to human cancer. Here, we report that JNK2 is activated in over 70% of human squamous cell carcinoma (SCC) samples and that inhibition of JNK2 pharmacologically or genetically impairs tumorigenesis of human SCC cells. Most importantly, JNK2, but not JNK1, is sufficient to couple with oncogenic Ras to transform primary human epidermal cells into malignancy with features of SCC. JNK2 prevents Ras-induced cell senescence and growth arrest by reducing the expression levels of the cell cycle inhibitor p16 and NF-κB activation. On the other hand, JNK, along with PI3K, is essential for Ras-induced glycolysis, an energy producing process known to benefit cancer growth. These data indicate that JNK2 collaborates with other oncogenes, such as Ras, at multiple molecular levels to promote tumorigenesis and hence represents a promising therapeutic target for cancer. PMID:20354187

  8. Tumor Necrosis Factor-α and Apoptosis Signal-Regulating Kinase 1 Control Reactive Oxygen Species Release, Mitochondrial Autophagy and C-Jun N-Terminal Kinase/P38 Phosphorylation During Necrotizing Enterocolitis

    Directory of Open Access Journals (Sweden)

    Naira Baregamian

    2009-01-01

    Full Text Available Background: Oxidative stress and inflammation may contribute to the disruption of the protective gut barrier through various mechanisms; mitochondrial dysfunction resulting from inflammatory and oxidative injury may potentially be a significant source of apoptosis during necrotizing enterocolitis (NEC. Tumor necrosis factor (TNFα is thought to generate reactive oxygen species (ROS and activate the apoptosis signal-regulating kinase 1 (ASK1-c-Jun N-terminal kinase (JNK/p38 pathway. Hence, the focus of our study was to examine the effects of TNFα/ROs on mitochondrial function, ASK1-JNK/p38 cascade activation in intestinal epithelial cells during NEC.

  9. Stress-induced phosphorylation of c-Jun-N-terminal kinases and nuclear translocation of Hsp70 in the Wistar rat hippocampus

    Directory of Open Access Journals (Sweden)

    Adžić M.

    2009-01-01

    Full Text Available Glucocorticoids are key regulators of the neuroendocrine stress response in the hippocampus. Their action is partly mediated through the subfamily of MAPKs termed c-Jun-N-terminal kinases (JNKs,whose activation correlates with neurodegeneration. The stress response also involves activation of cell protective mechanisms through various heat shock proteins (HSPs that mediate neuroprotection. We followed both JNKs and Hsp70 signals in the cytoplasmic and nuclear compartments of the hippocampus of Wistar male rats exposed to acute, chronic, and combined stress. The activity of JNK1 was decreased in both compartments by all three types of stress, while the activity of cytoplasmic JNK2/3 was elevated in acute and unaltered or lowered in chronic and combined stress. Under all stress conditions, Hsp70 translocation to the nucleus was markedly increased. The results suggest that neurodegenerative signaling of JNKs may be counteracted by increase of nuclear Hsp70,especially under chronic stress.

  10. Effect of Jun N-terminal kinase 1 and 2 on the replication of Penicillium marneffei in human macrophages.

    Science.gov (United States)

    Chen, Renqiong; Xi, Liyan; Huang, Xiaowen; Ma, Tuan; Ren, Hong; Ji, Guangquan

    2015-05-01

    Penicillium marneffei (P. marneffei) is a human pathogen which persists in macrophages and threatens the immunocompromised patients. To clarify the mechanisms involved, we evaluated the effect of c-Jun N-terminal kinase 1 and 2 (JNK1/2) on cytokine expression, phagosomal maturation and multiplication of P. marneffei in P. marneffei-stimulated human macrophages. P. marneffei induced the rapid phosphorylation of JNK1/2. Using the specific inhibitor of JNK1/2 (SP600125), we found that the inhibition of JNK1/2 suppressed P. marneffei-induced tumor necrosis factor-α and IL-10 production. In addition, the presence of SP600125 increased phagosomal acidification and maturation and decreased intracellular replication. These data suggest that JNK1/2 may play an important role in promoting the replication of P. marneffei. Our findings further indicate that the pathogen through the JNK1/2 pathway may attenuate the immune response and macrophage antifungal function.

  11. Analyses of Compact Trichinella Kinomes Reveal a MOS-Like Protein Kinase with a Unique N-Terminal Domain.

    Science.gov (United States)

    Stroehlein, Andreas J; Young, Neil D; Korhonen, Pasi K; Chang, Bill C H; Sternberg, Paul W; La Rosa, Giuseppe; Pozio, Edoardo; Gasser, Robin B

    2016-01-01

    Parasitic worms of the genus Trichinella (phylum Nematoda; class Enoplea) represent a complex of at least twelve taxa that infect a range of different host animals, including humans, around the world. They are foodborne, intracellular nematodes, and their life cycles differ substantially from those of other nematodes. The recent characterization of the genomes and transcriptomes of all twelve recognized taxa of Trichinella now allows, for the first time, detailed studies of their molecular biology. In the present study, we defined, curated, and compared the protein kinase complements (kinomes) of Trichinella spiralis and T. pseudospiralis using an integrated bioinformatic workflow employing transcriptomic and genomic data sets. We examined how variation in the kinome might link to unique aspects of Trichinella morphology, biology, and evolution. Furthermore, we utilized in silico structural modeling to discover and characterize a novel, MOS-like kinase with an unusual, previously undescribed N-terminal domain. Taken together, the present findings provide a basis for comparative investigations of nematode kinomes, and might facilitate the identification of Enoplea-specific intervention and diagnostic targets. Importantly, the in silico modeling approach assessed here provides an exciting prospect of being able to identify and classify currently unknown (orphan) kinases, as a foundation for their subsequent structural and functional investigation. PMID:27412987

  12. Analyses of Compact Trichinella Kinomes Reveal a MOS-Like Protein Kinase with a Unique N-Terminal Domain

    Directory of Open Access Journals (Sweden)

    Andreas J. Stroehlein

    2016-09-01

    Full Text Available Parasitic worms of the genus Trichinella (phylum Nematoda; class Enoplea represent a complex of at least twelve taxa that infect a range of different host animals, including humans, around the world. They are foodborne, intracellular nematodes, and their life cycles differ substantially from those of other nematodes. The recent characterization of the genomes and transcriptomes of all twelve recognized taxa of Trichinella now allows, for the first time, detailed studies of their molecular biology. In the present study, we defined, curated, and compared the protein kinase complements (kinomes of Trichinella spiralis and T. pseudospiralis using an integrated bioinformatic workflow employing transcriptomic and genomic data sets. We examined how variation in the kinome might link to unique aspects of Trichinella morphology, biology, and evolution. Furthermore, we utilized in silico structural modeling to discover and characterize a novel, MOS-like kinase with an unusual, previously undescribed N-terminal domain. Taken together, the present findings provide a basis for comparative investigations of nematode kinomes, and might facilitate the identification of Enoplea-specific intervention and diagnostic targets. Importantly, the in silico modeling approach assessed here provides an exciting prospect of being able to identify and classify currently unknown (orphan kinases, as a foundation for their subsequent structural and functional investigation.

  13. N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel

    Science.gov (United States)

    Hynkova, Anna; Marsakova, Lenka; Vaskova, Jana; Vlachova, Viktorie

    2016-06-01

    Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence.

  14. N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel.

    Science.gov (United States)

    Hynkova, Anna; Marsakova, Lenka; Vaskova, Jana; Vlachova, Viktorie

    2016-01-01

    Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence. PMID:27345869

  15. The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation.

    Science.gov (United States)

    Absmeier, Eva; Wollenhaupt, Jan; Mozaffari-Jovin, Sina; Becke, Christian; Lee, Chung-Tien; Preussner, Marco; Heyd, Florian; Urlaub, Henning; Lührmann, Reinhard; Santos, Karine F; Wahl, Markus C

    2015-12-15

    The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼ 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation. PMID:26637280

  16. Receptor binding and adenylate cyclase activities of glucagon analogues modified in the N-terminal region

    Energy Technology Data Exchange (ETDEWEB)

    McKee, R.L.; Pelton, J.T.; Trivedi, D.; Johnson, D.G.; Coy, D.H.; Sueiras-Diaz, J.; Hruby, V.J.

    1986-04-08

    In this study, we determined the ability of four N-terminally modified derivatives of glucagon, (3-Me-His1,Arg12)-, (Phe1,Arg12)-, (D-Ala4,Arg12)-, and (D-Phe4)glucagon, to compete with 125I-glucagon for binding sites specific for glucagon in hepatic plasma membranes and to activate the hepatic adenylate cyclase system, the second step involved in producing many of the physiological effects of glucagon. Relative to the native hormone, (3-Me-His1,Arg12)glucagon binds approximately twofold greater to hepatic plasma membranes but is fivefold less potent in the adenylate cyclase assay. (Phe1,Arg12)glucagon binds threefold weaker and is also approximately fivefold less potent in adenylate cyclase activity. In addition, both analogues are partial agonists with respect to adenylate cyclase. These results support the critical role of the N-terminal histidine residue in eliciting maximal transduction of the hormonal message. (D-Ala4,Arg12)glucagon and (D-Phe4)glucagon, analogues designed to examine the possible importance of a beta-bend conformation in the N-terminal region of glucagon for binding and biological activities, have binding potencies relative to glucagon of 31% and 69%, respectively. (D-Ala4,Arg12)glucagon is a partial agonist in the adenylate cyclase assay system having a fourfold reduction in potency, while the (D-Phe4) derivative is a full agonist essentially equipotent with the native hormone. These results do not necessarily support the role of an N-terminal beta-bend in glucagon receptor recognition. With respect to in vivo glycogenolysis activities, all of the analogues have previously been reported to be full agonists.

  17. Resveratrol alleviates diabetes-induced testicular dysfunction by inhibiting oxidative stress and c-Jun N-terminal kinase signaling in rats.

    Science.gov (United States)

    Faid, Iman; Al-Hussaini, Heba; Kilarkaje, Narayana

    2015-12-15

    Diabetes adversely affects reproductive functions in humans and animals. The present study investigated the effects of Resveratrol on diabetes-induced alterations in oxidative stress, c-Jun N-terminal kinase (JNK) signaling and apoptosis in the testis. Adult male Wistar rats (13-15 weeks; n=6/group) were segregated into 1) normal control, 2) Resveratrol-treated (5mg/kg; ip; given during last 3 weeks), 3) Streptozotocin-induced diabetic and, 4) Resveratrol-treated diabetic groups, and euthanized on day 42 after the confirmation of diabetes. Resveratrol did not normalize blood glucose levels in diabetic rats. Resveratrol supplementation recovered diabetes-induced decreases in reproductive organ weights, sperm count and motility, intra-testicular levels of superoxide dismutase, catalase, and glutathione peroxidase and an increase in 4-hydroxynonenal activities (Prats. These results suggest that Resveratrol supplementation may be a useful strategy to treat diabetes-induced testicular dysfunction. PMID:26499206

  18. EGCG-targeted p57/KIP2 reduces tumorigenicity of oral carcinoma cells: Role of c-Jun N-terminal kinase

    International Nuclear Information System (INIS)

    The green tea polyphenol epigallocatechin-3-gallate (EGCG) regulates gene expression differentially in tumor and normal cells. In normal human primary epidermal keratinocytes (NHEK), one of the key mediators of EGCG action is p57/KIP2, a cyclin-dependent kinase (CDK) inhibitor. EGCG potently induces p57 in NHEK, but not in epithelial cancer cells. In humans, reduced expression of p57 often is associated with advanced tumors, and tumor cells with inactivated p57 undergo apoptosis when exposed to EGCG. The mechanism of p57 induction by EGCG is not well understood. Here, we show that in NHEK, EGCG-induces p57 via the p38 mitogen-activated protein kinase (MAPK) signaling pathway. In p57-negative tumor cells, JNK signaling mediates EGCG-induced apoptosis, and exogenous expression of p57 suppresses EGCG-induced apoptosis via inhibition of c-Jun N-terminal kinase (JNK). We also found that restoration of p57 expression in tumor cells significantly reduced tumorigenicity in athymic mice. These results suggest that p57 expression may be an useful indicator for the clinical course of cancers, and could be potentially useful as a target for cancer therapies

  19. c-Jun N-terminal kinase is required for thermotherapy-induced apoptosis in human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Feng Xiao; Bin Liu; Qing-Xian Zhu

    2012-01-01

    AIM:To investigate the role of c-Jun N-terminal kinase (JNK) in thermotherapy-induced apoptosis in human gastric cancer SGC-7901 cells.METHODS:Human gastric cancer SGC-7901 cells were cultured in vitro.Following thermotherapy at 43 ℃ for 0,0.5,1,2 or 3 h,the cells were cultured for a further 24 h with or without the JNK specific inhibitor,SP600125 for 2 h.Apoptosis was evaluated by immunohistochemistry [terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)] and flow cytometry (Annexin vs propidium iodide).Cell proliferation was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.The production of p-JNK,Bcl-2,Bax and caspase-3 proteins was evaluated by Western blotting.The expression of JNK at mRNA level was determined by reverse transcription polymerase chain reaction.RESULTS:The Proliferation of gastric carcinoma SGC-7901 cells was significantly inhibited following thermotherapy,and was 32.7%,30.6%,43.8% and 52.9% at 0.5,1,2 and 3 h post-thermotherapy,respectively.Flow cytometry analysis revealed an increased population of SGC-7901 cells in G0/G1 phase,but a reduced population in S phase following therrnotherapy for 1 or 2 h,compared to untreated cells (P < 0.05).The increased number of SGC-7901 cells in G0/G1 phase was consistent with induced apoptosis (flow cytometry) following thermotherapy for 0.5,1,2 or 3 h,compared to the untreated group (46.5% ± 0.23%,39.9% ± 0.53%,56.6% ±0.35% and 50.4% ± 0.29% vs 7.3% ± 0.10%,P < 0.01),respectively.This was supported by the TUNEL assay (48.2% ± 0.4%,40.1% ± 0.2%,61.2% ± 0.29% and 52.0% ± 0.42% vs 12.2% ± 0.22%,P < 0.01) respectively.More importantly,the expression of p-JNK protein and JNK mRNA levels were significantly higher at 0.5 h than at 0 h post-treatment (P < 0.01),and peaked at 2 h.A similar pattem was detected for Bax and caspase-3 proteins.Bcl-2 increased at 0.5 h,peaked at 1 h,and then decreased

  20. C-Jun N-terminal kinase controls TDP-43 accumulation in stress granules induced by oxidative stress

    Directory of Open Access Journals (Sweden)

    Masters Colin L

    2011-08-01

    Full Text Available Abstract Background TDP-43 proteinopathies are characterized by loss of nuclear TDP-43 expression and formation of C-terminal TDP-43 fragmentation and accumulation in the cytoplasm. Recent studies have shown that TDP-43 can accumulate in RNA stress granules (SGs in response to cell stresses and this could be associated with subsequent formation of TDP-43 ubiquinated protein aggregates. However, the initial mechanisms controlling endogenous TDP-43 accumulation in SGs during chronic disease are not understood. In this study we investigated the mechanism of TDP-43 processing and accumulation in SGs in SH-SY5Y neuronal-like cells exposed to chronic oxidative stress. Cell cultures were treated overnight with the mitochondrial inhibitor paraquat and examined for TDP-43 and SG processing. Results We found that mild stress induced by paraquat led to formation of TDP-43 and HuR-positive SGs, a proportion of which were ubiquitinated. The co-localization of TDP-43 with SGs could be fully prevented by inhibition of c-Jun N-terminal kinase (JNK. JNK inhibition did not prevent formation of HuR-positive SGs and did not prevent diffuse TDP-43 accumulation in the cytosol. In contrast, ERK or p38 inhibition prevented formation of both TDP-43 and HuR-positive SGs. JNK inhibition also inhibited TDP-43 SG localization in cells acutely treated with sodium arsenite and reduced the number of aggregates per cell in cultures transfected with C-terminal TDP-43 162-414 and 219-414 constructs. Conclusions Our studies are the first to demonstrate a critical role for kinase control of TDP-43 accumulation in SGs and may have important implications for development of treatments for FTD and ALS, targeting cell signal pathway control of TDP-43 aggregation.

  1. Design, synthesis and aphicidal activity of N-terminal modified insect kinin analogs.

    Science.gov (United States)

    Zhang, Chuanliang; Qu, Yanyan; Wu, Xiaoqing; Song, Dunlun; Ling, Yun; Yang, Xinling

    2015-06-01

    The insect kinins are a class of multifunctional insect neuropeptides present in a diverse variety of insects. Insect kinin analogs showed multiple bioactivities, especially, the aphicidal activity. To find a biostable and bioactive insecticide candidate with simplified structure, a series of N-terminal modified insect kinin analogs was designed and synthesized based on the lead compound [Aib]-Phe-Phe-[Aib]-Trp-Gly-NH2. Their aphicidal activity against the soybean aphid Aphis glycines was evaluated. The results showed that all the analogs maintained the aphicidal activity. In particular, the aphicidal activity of the pentapeptide analog X Phe-Phe-[Aib]-Trp-Gly-NH2 (LC50=0.045mmol/L) was similar to the lead compound (LC50=0.048mmol/L). This indicated that the N-terminal protective group may not play an important role in the activity and the analogs structure could be simplified to pentapeptide analogs while retaining good aphicidal activity. The core pentapeptide analog X can be used as the lead compound for further chemical modifications to discover potential insecticides. PMID:25116632

  2. Myocardial protective effects of a c-Jun N-terminal kinase inhibitor in rats with brain death.

    Science.gov (United States)

    Guo, Wenzhi; Cao, Shengli; Yan, Bing; Zhang, Gong; Li, Jie; Zhao, Yongfu; Zhang, Shuijun

    2016-07-01

    To investigate whether the mitochondrial apoptotic pathway mediates myocardial cell injuries in rats under brain death (BD), and observe the effects and mechanisms of the c-Jun N-terminal kinase (JNK) inhibitor SP600125 on cell death in the heart. Forty healthy male Sprague-Dawley (SD) rats were randomized into four groups: sham group (dural external catheter with no BD); BD group (maintain the induced BD state for 6 hrs); BD + SP600125 group (intraperitoneal injection of SP600125 10 mg/kg 1 hr before inducing BD, and maintain BD for 6 hrs); and BD + Dimethyl Sulphoxide (DMSO) group (intraperitoneal injection of DMSO 1 hr before inducing BD, and maintain BD for 6 hrs). Real-time quantitative PCR was used to evaluate mRNA levels of Cyt-c and caspase-3. Western blot analysis was performed to examine the levels of mitochondrial apoptosis-related proteins p-JNK, Bcl-2, Bax, Cyt-c and Caspase-3. TUNEL assay was employed to evaluate myocardial apoptosis. Compared with the sham group, the BD group exhibited increased mitochondrial apoptosis-related gene expression, accompanied by the elevation of p-JNK expression and myocardial apoptosis. As the vehicle control, DMSO had no treatment effects. The BD + SP600125 group had decreased p-JNK expression, and reduced mitochondrial apoptosis-related gene expression. Furthermore, the apoptosis rate of myocardial cells was reduced. The JNK inhibitor SP600125 could protect myocardial cells under BD through the inhibition of mitochondrial apoptosis-related pathways. PMID:27072084

  3. Recombinant N-Terminal Slit2 Inhibits TGF-β-Induced Fibroblast Activation and Renal Fibrosis.

    Science.gov (United States)

    Yuen, Darren A; Huang, Yi-Wei; Liu, Guang-Ying; Patel, Sajedabanu; Fang, Fei; Zhou, Joyce; Thai, Kerri; Sidiqi, Ahmad; Szeto, Stephen G; Chan, Lauren; Lu, Mingliang; He, Xiaolin; John, Rohan; Gilbert, Richard E; Scholey, James W; Robinson, Lisa A

    2016-09-01

    Fibrosis and inflammation are closely intertwined injury pathways present in nearly all forms of CKD for which few safe and effective therapies exist. Slit glycoproteins signaling through Roundabout (Robo) receptors have been described to have anti-inflammatory effects through regulation of leukocyte cytoskeletal organization. Notably, cytoskeletal reorganization is also required for fibroblast responses to TGF-β Here, we examined whether Slit2 also controls TGF-β-induced renal fibrosis. In cultured renal fibroblasts, which we found to express Slit2 and Robo-1, the bioactive N-terminal fragment of Slit2 inhibited TGF-β-induced collagen synthesis, actin cytoskeletal reorganization, and Smad2/3 transcriptional activity, but the inactive C-terminal fragment of Slit2 did not. In mouse models of postischemic renal fibrosis and obstructive uropathy, treatment with N-terminal Slit2 before or after injury inhibited the development of renal fibrosis and preserved renal function, whereas the C-terminal Slit2 had no effect. Our data suggest that administration of recombinant Slit2 may be a new treatment strategy to arrest chronic injury progression after ischemic and obstructive renal insults by not only attenuating inflammation but also, directly inhibiting renal fibrosis.

  4. Colchicine induces apoptosis in HT‑29 human colon cancer cells via the AKT and c-Jun N-terminal kinase signaling pathways.

    Science.gov (United States)

    Huang, Zhen; Xu, Ye; Peng, Wei

    2015-10-01

    Colchicine is a natural compound, which belongs to the botanical family Colchicaceae and prevents growth of cancer cells via antimitotic activity by interacting with microtubules. Although numerous studies have demonstrated that the effect of colchicine on cell apoptosis is mediated by the activation of caspase‑3, the signaling pathways involved in the process remain unknown. In the current study, evidence is presented regarding the missing information using HT‑29 human colon cancer cells. The effect of colchicine on apoptosis in HT‑29 cells and the apoptosis‑associated signaling pathways were determined using various methods, including cell viability assay, Annexin V/propidium idodide (PI) binding, PI staining, Hoechst 33342 staining, mitochondrial membrane potential (Δψm) assay, reactive oxygen species (ROS) assay and western blot analysis. Colchicine was observed to induce a dose‑dependent reduction in cell viability in HT‑29 cells and early apoptosis occurred when the cells were treated with 1 µg/ml colchicine. Furthermore, colchicine treatment induced a loss of Δψm, increased ROS production, activated caspase‑3, upregulated BAX expression and downregulated Bcl‑2 expression, which evidenced the colchicine activity on apoptosis, potentially by acting via the intrinsic apoptotic signaling pathway. Colchicine increased phosphorylation of p38, although not phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase, which indicates that colchicine activates the p38 signaling pathway in order to induce cell apoptosis. Therefore, colchicine exhibited significant growth inhibition of the HT‑29 colon cancer cell line and induced apoptosis in the cells via the mitochondrial pathway, which is regulated by p38 signaling pathways. PMID:26299305

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

    Science.gov (United States)

    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. PMID:25510970

  6. N-Terminal methionine processing by the zinc-activated Plasmodium falciparum methionine aminopeptidase 1b.

    Science.gov (United States)

    Calcagno, Sarah; Klein, Christian D

    2016-08-01

    The methionine aminopeptidase 1b from Plasmodium falciparum (PfMetAP 1b) was cloned, expressed in Escherichia coli and characterized. Surprisingly, and in contrast to other methionine aminopeptidases (MetAPs) that require heavy-metal cofactors such as cobalt, the enzyme is reliably activated by zinc ions. Immobilization of the enzyme is possible by His-tag metal chelation to iminodiacetic acid-agarose and by covalent binding to chloroacetamido-hexyl-agarose. The covalently immobilized enzyme shows long-term stability, allowing a continuous, heterogenous processing of N-terminal methionines, for example, in recombinant proteins. Activation by zinc, instead of cobalt as for other MetAPs, avoids the introduction of heavy metals with toxicological liabilities and oxidative potential into biotechnological processes. The PfMetAP 1b therefore represents a useful tool for the enzymatic, posttranslational processing of recombinant proteins. PMID:27023914

  7. Antimicrobial activity of human prion protein is mediated by its N-terminal region.

    Directory of Open Access Journals (Sweden)

    Mukesh Pasupuleti

    Full Text Available BACKGROUND: Cellular prion-related protein (PrP(c is a cell-surface protein that is ubiquitously expressed in the human body. The multifunctionality of PrP(c, and presence of an exposed cationic and heparin-binding N-terminus, a feature characterizing many antimicrobial peptides, made us hypothesize that PrP(c could exert antimicrobial activity. METHODOLOGY AND PRINCIPAL FINDINGS: Intact recombinant PrP exerted antibacterial and antifungal effects at normal and low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide 20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein. Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the "classical" human antimicrobial peptide LL-37. In contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively charged (bacteria-mimicking liposomes. PrP furthermore showed an inducible expression during wounding of human skin ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-alpha in vitro. CONCLUSIONS: The demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could have a previously undisclosed role in host defense.

  8. Tumor suppressor BLU inhibits proliferation of nasopharyngeal carcinoma cells by regulation of cell cycle, c-Jun N-terminal kinase and the cyclin D1 promoter

    Directory of Open Access Journals (Sweden)

    Zhang Xiangning

    2012-06-01

    Full Text Available Abstract Background Tumor suppressor genes function to regulate and block tumor cell proliferation. To explore the mechanisms underlying the tumor suppression of BLU/ZMYND10 gene on a frequently lost human chromosomal region, an adenoviral vector with BLU cDNA insert was constructed. Methods BLU was re-expressed in nasopharyngeal carcinoma cells by transfection or viral infection. Clonogenic growth was assayed; cell cycle was analyzed by flow cytometry-based DNA content detection; c-Jun N-terminal kinase (JNK and cyclin D1 promoter activities were measured by reporter gene assay, and phosphorylation was measured by immunoblotting. The data for each pair of groups were compared with Student t tests. Results BLU inhibits clonogenic growth of nasopharyngeal carcinoma cells, arrests cell cycle at G1 phase, downregulates JNK and cyclin D1 promoter activities, and inhibits phosphorylation of c-Jun. Conclusions BLU inhibits growth of nasopharyngeal carcinoma cells by regulation of the JNK-cyclin D1 axis to exert tumor suppression.

  9. SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling in commercial and wild-type turkey leukocytes

    Science.gov (United States)

    Studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases (PTK) and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on days...

  10. Drosophila endocytic neoplastic tumor suppressor genes regulate Sav/Wts/Hpo signaling and the c-Jun N-terminal kinase pathway.

    Science.gov (United States)

    Robinson, Brian S; Moberg, Kenneth H

    2011-12-01

    Genetic screens in the fruit fly Drosophila melanogaster have identified a class of neoplastic tumor suppressor genes (endocytic nTSGs), which encode proteins that localize to endosomes and facilitate the trafficking of membrane-bound receptors and adhesion molecules into the degradative lysosome. Loss of endocytic nTSGs transforms imaginal disc epithelia into highly proliferative, invasive tissues that fail to differentiate and display defects in cellular apicobasal polarity, adhesion and tissue architecture. As vertebrate homologs of some Drosophila nTSGs are linked to tumor formation, identifying molecular changes in signaling associated with nTSG loss could inform understanding of neoplastic transformation in vertebrates. Here we show that mutations in genes that act at multiple steps of the endolysosomal pathway lead to autonomous activation of the Sav/Wts/Hpo (SWH) transcriptional effector Yki (YAP/TAZ in vertebrates) and the Jun N-terminal kinase (JNK), which is known to promote Yki activity in cells with disrupted polarity. Yki and JNK activity are elevated by mutations at multiple steps in the endolysosomal pathway including mutations in the AP-2σ gene, which encodes a component of the AP-2 adaptor complex that recruits cargoes into clathrin-coated pits for subsequent internalization. Moreover, reduction of JNK activity can decrease elevated Yki-signaling caused by altered endocytosis. These studies reveal a broad requirement for components of the endocytic pathway in regulating SWH and JNK outputs, and place Drosophila endocytic nTSGs into a network that involving two major signaling pathways implicated in oncogenesis. PMID:22101275

  11. Apoptosis induced by genipin in human leukemia K562 cells:involvement of C-Jun N-terminal kinase in G2/M arrest

    Institute of Scientific and Technical Information of China (English)

    Qian FENG; Hou-li CAO; Wei XU; Xiao-rong LI; Yan-qin REN; Lin-fang DU

    2011-01-01

    Aim:To investigate the effect of genipin on apoptosis in human leukemia K562 cells in vitro and elucidate the underlying mechanisms.Methods:The effect of genipin on K562 cell viability was measured using trypan blue dye exclusion and cell counting.Morphological changes were detected using phase-contrast microscopy.Apoptosis was analyzed using DNA ladder, propidium iodide(PI)-labeled flow cytometry(FCM)and Hoechst 33258 staining.The infiuence of genipin on cell cycle distribution was determined using Plstaining.Caspase 3 activity was analyzed to detect apoptosis at different time points.Protein levels of phospho-c-Jun,phosphor-C-Jun N-terminal kinase(p-JNK).phosphor-p38-Fas-L,p63,and Bax and the release of cytochrome c were detected using Western blot analysis.Results:Genipin reduced the viability of K562 cells with an IC50 value of approximately 250 μmol/L.Genipin 200-400 μmol/L induced formation of typicaI apoptotic bodies and DNA fragmentation.Additionally,genipin 400 μmol/L significantly increased the caspase 3activity from 8-24 h and arrested the cells in the G2/M phase.After stimulation with genipin 500 μmol/L, the levels of p-JNK, p-c-Jun.Fas-L,Bax.and cytochrome c were remarkably upregulated,but there were no obvious changes of p-p38.Genipin 200-500 μmol/Lsignificantly upregulated the Fas-L expression and downregulated p63 expression.Dicoumarol 100 μmol/L.a JNK1/2 inhibitor,markedly suppressed the formation of apoptotic bodies and JNK activation induced by genipin 400 μmol/L.Conclusion:These results suggest that genipin inhibits the proliferation of K562 cells and induces apoptosis through the activation of JNK and induction of the Fas ligand.

  12. Fine tuning of the catalytic activity of colicin e7 nuclease domain by systematic n-terminal mutations

    DEFF Research Database (Denmark)

    Németh, Eszter; Körtvélyesi, Tamás; Thulstrup, Peter W.;

    2014-01-01

    The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C-terminal HNH motif. Interestingly, the deletion of four N-terminal residues (446–449NColE75KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuc...

  13. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

    Energy Technology Data Exchange (ETDEWEB)

    Gottipati, Keerthi; Acholi, Sudheer [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States); Ruggli, Nicolas [Institute of Virology and Immunology, CH-3147 Mittelhäusern (Switzerland); Choi, Kyung H., E-mail: kychoi@utmb.edu [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States)

    2014-03-15

    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 that 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.

  14. Promoter-dependent activity on androgen receptor N-terminal domain mutations in androgen insensitivity syndrome.

    Science.gov (United States)

    Tadokoro-Cuccaro, Rieko; Davies, John; Mongan, Nigel P; Bunch, Trevor; Brown, Rosalind S; Audi, Laura; Watt, Kate; McEwan, Iain J; Hughes, Ieuan A

    2014-01-01

    Androgen receptor (AR) mutations are associated with androgen insensitivity syndrome (AIS). Missense mutations identified in the AR-N-terminal domain (AR-NTD) are rare, and clinical phenotypes are typically mild. We investigated 7 missense mutations and 2 insertion/deletions located in the AR-NTD. This study aimed to elucidate the pathogenic role of AR-NTD mutants in AIS and to use this knowledge to further define AR-NTD function. AR-NTD mutations (Q120E, A159T, G216R, N235K, G248V, L272F, and P380R) were introduced into AR-expression plasmids. Stably expressing cell lines were established for del57L and ins58L. Transactivation was measured using luciferase reporter constructs under the control of GRE and Pem promoters. Intrinsic fluorescence spectroscopy and partial proteolysis studies were performed for mutations which showed reduced activities by using a purified AR-AF1 protein. Pem-luciferase reporter activation was reduced for A159T, N235K, and G248V but not the GRE-luciferase reporter. Protein structure analysis detected no significant change in the AR-AF1 region for these mutations. Reduced cellular expression and transactivation activity were observed for ins58L. The mutations Q120E, G216R, L272F, P380R, and del57L showed small or no detectable changes in function. Thus, clinical and experimental analyses have identified novel AR-signalling defects associated with mutations in the structurally disordered AR-NTD domain in patients with AIS.

  15. Involvement of c-Jun N-terminal kinase in reversal of multidrug resistance of human leukemia cells in hypoxia by 5-bromotetrandrine.

    Science.gov (United States)

    Zhang, Wei; Chen, Bao-an; Jin, Jun-fei; He, You-ji; Niu, Yi-qi

    2013-11-01

    5-Bromotetrandrine (BrTet), a candidate multidrug resistance (MDR) modulator, is a potential compound for use in cancer therapy when combined with anticancer agents such as daunorubicin (DNR) and paclitaxel. The purposeof this study was to investigate the mechanism of reversal of P-glycoprotein (P-gp)-mediated MDR by BrTet and the involvement of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway in both adriamycin-sensitive K562 and adriamycin-resistant K562 (KA) leukemia cells in hypoxia. The combination of BrTet and DNR decreased both phosphorylated JNK1/2 and MDR1/P-gp levels under hypoxic conditions. Furthermore, a pharmacological inhibitor of JNK, SP600125, or small interfering RNA (siRNA) oligonucleotides to both JNK1 and JNK2 reversed BrTet- or DNR-induced JNK phosphorylation and MDR1/P-gp levels. We further demonstrated that the decreased JNK phosphorylation and MDR1/P-gp levels were associated with a significant increase in intracellular accumulation of DNR, which dramatically enhanced the sensitivity of drug-resistant KA cells to DNR, and led to cellular apoptosis through activation of the caspase-3 pathway. It is concluded that using BrTet in combination with other chemotherapeutic agents and pharmacological inhibitors of JNK can abrogate the P-gp-induced MDR in adriamycin-resistant K562 cells, which has potential clinical relevance in cancer therapy for chemotherapeutic-resistant human leukemia. PMID:23418897

  16. HSF1/HSP70通路抑制c-Jun氨基末端激酶的活化保护UVA诱导的HaCaT细胞凋亡%Protection of HSF1/HSP70 pathway on UVA-induced HaCaT cells apoptosis via inhibiting the activation of c-Jun N-terminal kinase

    Institute of Scientific and Technical Information of China (English)

    王晓雯; 王春波; 李丙华; 韩彦弢

    2012-01-01

    目的 观察热休克转录因子1( HSF1)与热休克蛋白70( HSP70)对紫外线A(UVA)诱导HaCaT细胞凋亡的保护作用及其机制.方法 建立8mJ/cm2 UVA辐射损伤HaCaT细胞的病理模型.将细胞随机分为对照组、8mJ/cm2 UVA照射组、HSP70转录抑制剂组(50 μmol/L槲皮素).Honechst 33258荧光染色观察细胞凋亡;蛋白质印迹法检测UVA辐射HaCaT细胞后p-HSF1和HSP70蛋白的经时变化及UVA辐射后孵育6h JNK(c-Jun氨基末端激酶)、p-JNK的蛋白表达;Real-Time PCR检测HSP70 mRNA的表达.结果 UVA辐射后HaCaT细胞内p-HSF1、HSP70蛋白表达量均出现先增加后减少的时间依赖性趋势,其中p-HSF1于lh开始增加,3h达高峰,HSP70于6h达高峰,24h基本恢复原始水平;UVA辐射前预先加入HSP70转录抑制剂槲皮素能显著抑制HSP70 mRNA的表达,增加p-JNK的表达量,同时Honechst 33258荧光染色观察其与UVA辐射组比较凋亡率明显升高.结论 8mJ/cm2 UVA辐射HaCaT细胞在一定时间内可使HSF1活化致HSP70表达增加.HSFl/HSP70通路对UVA诱导的HaCaT细胞凋亡具有保护作用,其机制与HSP70大量表达后抑制JNK的活化有关.%Objective To investigate the protective effect of heat shock factorl ( HSF1) and heat shock protein70 ( HSP70) on ultraviolet A ( UVA ) -induced HaCaT cells apoptosis and its mechanism. Methods The apoptotic HaCaT cell model was induced by UVA irradiation (8mJ/cm ). The cells were randomly divided into three groups, including a control group, a model group (8mJ/cm UVA) and a HSP70 transcription inhibitor group (50 μmol/L quercetin). The morphologic alteration of apoptotic cells was investigated by using Hoechst 33258 fluorescent staining. Western blotting was used to investigate protein expression levels of phosphorylated HSF1 and HSP70 at different time points, as well as c-Jun N-terminal kinase ( JNK ) andphosphorylated JNK were investigated after incubating for 6 hours following UVA irradiation. HSP70 mRNA was

  17. Diallyl trisulfide-induced apoptosis in human prostate cancer cells involves c-Jun N-terminal kinase and extracellular-signal regulated kinase-mediated phosphorylation of Bcl-2.

    Science.gov (United States)

    Xiao, Dong; Choi, Sunga; Johnson, Daniel E; Vogel, Victor G; Johnson, Candace S; Trump, Donald L; Lee, Yong J; Singh, Shivendra V

    2004-07-22

    Garlic-derived organosulfides (OSCs) including diallyl trisulfide (DATS) are highly effective in affording protection against chemically induced cancer in animals. Evidence is also mounting to indicate that some naturally occurring OSCs can suppress proliferation of cancer cells by causing apoptosis, but the sequence of events leading to proapoptotic effect of OSCs is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate that DATS is a significantly more potent apoptosis inducer than diallyl sulfide (DAS) or diallyl disulfide (DADS). DATS-induced apoptosis in PC-3 cells was associated with phosphorylation of Bcl-2, reduced Bcl-2 : Bax interaction, and cleavage of procaspase-9 and -3. Bcl-2 overexpressing PC-3 cells were significantly more resistant to apoptosis induction by DATS compared with vector-transfected control cells. DATS treatment resulted in activation of extracellular-signal regulated kinase 1/2 (ERK1/2) and c-jun N-terminal kinase 1 (JNK1) and/or JNK2, but not p38 mitogen-activated protein kinase. Phosphorylation of Bcl-2 in DATS-treated PC-3 cells was fully blocked in the presence of JNK-specific inhibitor SP600125. Moreover, JNK inhibitor afforded significant protection against DATS-induced apoptosis in both cells. DATS-induced Bcl-2 phosphorylation and apoptosis were partially attenuated by pharmacological inhibition of ERK1/2 using PD98059 or U0126. Overexpression of catalase inhibited DATS-mediated activation of JNK1/2, but not ERK1/2, and apoptosis induction in DU145 cells suggesting involvement of hydrogen peroxide as a second messenger in DATS-induced apoptosis. In conclusion, our data point towards important roles for Bcl-2, JNK and ERK in DATS-induced apoptosis in human prostate cancer cells.

  18. 90-kDa ribosomal S6 kinase is phosphorylated and activated by 3-phosphoinositide-dependent protein kinase-1

    DEFF Research Database (Denmark)

    Jensen, Claus Antonio Juel; Buch, M B; Krag, T O;

    1999-01-01

    90-kDa ribosomal S6 kinase-2 (RSK2) belongs to a family of growth factor-activated serine/threonine kinases composed of two kinase domains connected by a regulatory linker region. The N-terminal kinase of RSK2 is involved in substrate phosphorylation. Its activation requires phosphorylation of th...... of Ser(227), Ser(369), and Ser(386). Our study extend recent findings which implicate PDK1 in the activation of protein kinases B and C and p70(S6K), suggesting that PDK1 controls several major growth factor-activated signal transduction pathways.......90-kDa ribosomal S6 kinase-2 (RSK2) belongs to a family of growth factor-activated serine/threonine kinases composed of two kinase domains connected by a regulatory linker region. The N-terminal kinase of RSK2 is involved in substrate phosphorylation. Its activation requires phosphorylation...... of the linker region at Ser(369), catalyzed by extracellular signal-regulated kinase (ERK), and at Ser(386), catalyzed by the C-terminal kinase, after its activation by ERK. In addition, the N-terminal kinase must be phosphorylated at Ser(227) in the activation loop by an as yet unidentified kinase. Here, we...

  19. N-terminal aromatic residues closely impact the cytolytic activity of cupiennin 1a, a major spider venom peptide.

    Science.gov (United States)

    Kuhn-Nentwig, Lucia; Sheynis, Tania; Kolusheva, Sofiya; Nentwig, Wolfgang; Jelinek, Raz

    2013-12-01

    Cupiennins are small cationic α-helical peptides from the venom of the ctenid spider Cupiennius salei which are characterized by high bactericidal as well as hemolytic activities. To gain insight into the determinants responsible for the broad cytolytic activities, two analogues of cupiennin 1a with different N-terminal hydrophobicities were designed. The insecticidal, bactericidal and hemolytic activities of these analogues were assayed and compared to the native peptide. Specifically, substitution of two N-terminal Phe residues by Ala results in less pronounced insecticidal and cytolytic activity, whereas a substitution by Lys reduces strongly its bactericidal activity and completely diminishes its hemolytic activity up to very high tested concentrations. Biophysical analyses of peptide/bilayer membrane interactions point to distinct interactions of the analogues with lipid bilayers, and dependence upon membrane surface charge. Indeed, we find that lower hemolytic activity was correlated with less surface association of the analogues. In contrast, our data indicate that the reduced bactericidal activity of the two cupiennin 1a analogues likely correspond to greater bilayer-surface localization of the peptides. Overall, ultimate insertion and destruction of the host cell membrane is highly dependent on the presence of Phe-2 and Phe-6 (Cu 1a) or Leu-6 (Cu 2a) in the N-terminal sequences of native cupiennins.

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

    International Nuclear Information System (INIS)

    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. 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 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 critical

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

    Energy Technology Data Exchange (ETDEWEB)

    Du, Kuo; Williams, C. David; McGill, Mitchell R.; Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu

    2014-11-15

    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. 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 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 critical

  2. Hyperoside Downregulates the Receptor for Advanced Glycation End Products (RAGE and Promotes Proliferation in ECV304 Cells via the c-Jun N-Terminal Kinases (JNK Pathway Following Stimulation by Advanced Glycation End-Products In Vitro

    Directory of Open Access Journals (Sweden)

    Zhengyu Zhang

    2013-11-01

    Full Text Available Hyperoside is a major active constituent in many medicinal plants which are traditionally used in Chinese medicines for their neuroprotective, anti-inflammatory and antioxidative effects. The molecular mechanisms underlying these effects are unknown. In this study, quiescent ECV304 cells were treated in vitro with advanced glycation end products (AGEs in the presence or absence of hyperoside. The results demonstrated that AGEs induced c-Jun N-terminal kinases (JNK activation and apoptosis in ECV304 cells. Hyperoside inhibited these effects and promoted ECV304 cell proliferation. Furthermore, hyperoside significantly inhibited RAGE expression in AGE-stimulated ECV304 cells, whereas knockdown of RAGE inhibited AGE-induced JNK activation. These results suggested that AGEs may promote JNK activation, leading to viability inhibition of ECV304 cells via the RAGE signaling pathway. These effects could be inhibited by hyperoside. Our findings suggest a novel role for hyperoside in the treatment and prevention of diabetes.

  3. c-Jun N-terminal kinase 3 expression in the retina of ocular hypertension mice: a possible target to reduce ganglion cell apoptosis

    Directory of Open Access Journals (Sweden)

    Yue He

    2015-01-01

    Full Text Available Glaucoma, a type of optic neuropathy, is characterized by the loss of retinal ganglion cells. It remains controversial whether c-Jun N-terminal kinase (JNK participates in the apoptosis of retinal ganglion cells in glaucoma. This study sought to explore a possible mechanism of action of JNK signaling pathway in glaucoma-induced retinal optic nerve damage. We established a mouse model of chronic ocular hypertension by reducing the aqueous humor followed by photocoagulation using the laser ignition method. Results showed significant pathological changes in the ocular tissues after the injury. Apoptosis of retinal ganglion cells increased with increased intraocular pressure, as did JNK3 mRNA expression in the retina. These data indicated that the increased expression of JNK3 mRNA was strongly associated with the increase in intraocular pressure in the retina, and correlated positively with the apoptosis of retinal ganglion cells.

  4. c-Jun N-terminal kinase 3 expression in the retina of ocular hypertension mice:a possible target to reduce ganglion cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Yue He; Jie Chen; Shu-guang Zhang; Yuan-sheng Yuan; Yan Li; Hong-bin Lv; Jin-hua Gan

    2015-01-01

    Glaucoma, a type of optic neuropathy, is characterized by the loss of retinal ganglion cells. It remains controversial whether c-Jun N-terminal kinase (JNK) participates in the apoptosis of retinal ganglion cells in glaucoma. This study sought to explore a possible mechanism of action of JNK signaling pathway in glaucoma-induced retinal optic nerve damage. We established a mouse model of chronic ocular hypertension by reducing the aqueous humor followed by pho-tocoagulation using the laser ignition method. Results showed signiifcant pathological changes in the ocular tissues after the injury. Apoptosis of retinal ganglion cells increased with increased intraocular pressure, as did JNK3 mRNA expression in the retina. These data indicated that the increased expression of JNK3 mRNA was strongly associated with the increase in intraocular pressure in the retina, and correlated positively with the apoptosis of retinal ganglion cells.

  5. Effect of N-Terminal Acylation on the Activity of Myostatin Inhibitory Peptides.

    Science.gov (United States)

    Takayama, Kentaro; Nakamura, Akari; Rentier, Cédric; Mino, Yusaku; Asari, Tomo; Saga, Yusuke; Taguchi, Akihiro; Yakushiji, Fumika; Hayashi, Yoshio

    2016-04-19

    Inhibition of myostatin, which negatively regulates skeletal muscle growth, is a promising strategy for the treatment of muscle atrophic disorders, such as muscular dystrophy, cachexia and sarcopenia. Recently, we identified peptide A (H-WRQNTRYSRIEAIKIQILSKLRL-NH2 ), the 23-amino-acid minimum myostatin inhibitory peptide derived from mouse myostatin prodomain, and highlighted the importance of its N-terminal tryptophan residue for the effective inhibition. In this study, we synthesized a series of acylated peptide derivatives focused on the tryptophan residue to develop potent myostatin inhibitors. As a result of the investigation, a more potent derivative of peptide A was successfully identified in which the N-terminal tryptophan residue is replaced with a 2-naphthyloxyacetyl moiety to give an inhibitory peptide three times (1.19±0.11 μm) more potent than parent peptide A (3.53±0.25 μm). This peptide could prove useful as a new starting point for the development of improved inhibitory peptides. PMID:26954624

  6. Mitogen-activated protein kinases mediate Mycobacterium tuberculosis–induced CD44 surface expression in monocytes

    Indian Academy of Sciences (India)

    Natarajan Palaniappan; S Anbalagan; Sujatha Narayanan

    2012-03-01

    CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are involved in M. tuberculosis–induced CD44 surface expression in monocytic cells are currently unknown. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv and H37Ra induced distinct, time-dependent, phosphorylation of mitogen-activated protein kinase kinase-1, extracellular signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, p38 mitogen-activated protein kinase and c-jun N-terminal kinases. The strains also differed in their usage of CD14 and human leukocyte antigen-DR (HLA-DR) receptors in mediating mitogen-activated protein kinase activation. M. tuberculosis H37Rv strain induced lower CD44 surface expression and tumour necrosis factor-alpha levels, whereas H37Ra the reverse. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and c-jun N-terminal kinase, we report that inhibition of extracellular signal regulated kinase 1/2 and c-jun N-terminal kinases increases, but that inhibition of p38 mitogen-activated protein kinase decreases M. tuberculosis–induced CD44 surface expression in THP-1 human monocytes.

  7. Mitogen-activated protein kinases in atherosclerosis

    Directory of Open Access Journals (Sweden)

    Dorota Bryk

    2014-01-01

    Full Text Available Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase, JNK (c-Jun N-terminal kinase and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis.

  8. Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain.

    Directory of Open Access Journals (Sweden)

    Kepa B Uribe

    Full Text Available Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active "soluble AC". The calpain-mediated ACT processing allows trafficking of the "soluble AC" domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP "pools", which would play different roles in the cell pathophysiology.

  9. Allosteric regulation of protein kinase PKCζ by the N-terminal C1 domain and small compounds to the PIF-pocket

    DEFF Research Database (Denmark)

    Lopez-Garcia, Laura A; Schulze, Jörg O; Fröhner, Wolfgang;

    2011-01-01

    Protein kinases are key mediators of cellular signaling, and therefore, their activities are tightly controlled. AGC kinases are regulated by phosphorylation and by N- and C-terminal regions. Here, we studied the molecular mechanism of inhibition of atypical PKCζ and found that the inhibition by ...

  10. Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration.

    Science.gov (United States)

    Hayashi, Moyuru; Tamura, Yosuke; Dohmae, Naoshi; Kojima, Soichi; Shimonaka, Motoyuki

    2008-05-01

    Angiostatin, a potent inhibitor of angiogenesis, is derived from the fibrinolytic proenzyme, plasminogen, by enzymatic processing. Plasminogen N-terminal activation peptide (PAP) is one of the products concomitantly released aside from angiostatin (kringles 1-4) and mini-plasminogen (kringle 5 plus the catalytic domain) when plasminogen is processed. To determine whether PAP alone or together with the angiostatin-related peptides derived from the processing of plasminogen modulate the proliferation and motility of endothelial cells, we have generated a recombinant PAP and used it to study its effects on endothelial cells in the presence and absence of the angiostatin-related peptides. Our results showed that PAP alone slightly increased the migration but not the proliferation of endothelial cells. However, in the presence of the angiostatin-related peptides, PAP attenuated the inhibitory activity of the angiostatin-related peptides on the proliferation and migration of endothelial cells. The inhibitory effect of PAP on the angiostatin-related peptides could be due to its binding to the kringle domains of the latter peptides. PMID:18294956

  11. Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration

    International Nuclear Information System (INIS)

    Angiostatin, a potent inhibitor of angiogenesis, is derived from the fibrinolytic proenzyme, plasminogen, by enzymatic processing. Plasminogen N-terminal activation peptide (PAP) is one of the products concomitantly released aside from angiostatin (kringles 1-4) and mini-plasminogen (kringle 5 plus the catalytic domain) when plasminogen is processed. To determine whether PAP alone or together with the angiostatin-related peptides derived from the processing of plasminogen modulate the proliferation and motility of endothelial cells, we have generated a recombinant PAP and used it to study its effects on endothelial cells in the presence and absence of the angiostatin-related peptides. Our results showed that PAP alone slightly increased the migration but not the proliferation of endothelial cells. However, in the presence of the angiostatin-related peptides, PAP attenuated the inhibitory activity of the angiostatin-related peptides on the proliferation and migration of endothelial cells. The inhibitory effect of PAP on the angiostatin-related peptides could be due to its binding to the kringle domains of the latter peptides

  12. Fast closure of N-terminal long loops but slow formation of β strands precedes the folding transition state of Escherichia coli adenylate kinase.

    Science.gov (United States)

    Orevi, Tomer; Ben Ishay, Eldad; Gershanov, Sivan Levin; Dalak, Mayan Ben; Amir, Dan; Haas, Elisha

    2014-05-20

    The nature of the earliest steps of the initiation of the folding pathway of globular proteins is still controversial. To elucidate the role of early closure of long loop structures in the folding transition, we studied the folding kinetics of subdomain structures in Escherichia coli adenylate kinase (AK) using Förster type resonance excitation energy transfer (FRET)-based methods. The overall folding rate of the AK molecule and of several segments that form native β strands is 0.5 ± 0.3 s(-1), in sharp contrast to the 1000-fold faster closure of three long loop structures in the CORE domain. A FRET-based "double kinetics" analysis revealed complex transient changes in the initially closed N-terminal loop structure that then opens and closes again at the end of the folding pathway. The study of subdomain folding in situ suggests a hierarchic ordered folding mechanism, in which early and rapid cross-linking by hydrophobic loop closure provides structural stabilization at the initiation of the folding pathway. PMID:24787383

  13. c-Jun N-terminal kinase phosphorylation of MARCKSL1 determines actin stability and migration in neurons and in cancer cells.

    Science.gov (United States)

    Björkblom, Benny; Padzik, Artur; Mohammad, Hasan; Westerlund, Nina; Komulainen, Emilia; Hollos, Patrik; Parviainen, Lotta; Papageorgiou, Anastassios C; Iljin, Kristiina; Kallioniemi, Olli; Kallajoki, Markku; Courtney, Michael J; Mågård, Mats; James, Peter; Coffey, Eleanor T

    2012-09-01

    Cell migration is a fundamental biological function, critical during development and regeneration, whereas deregulated migration underlies neurological birth defects and cancer metastasis. MARCKS-like protein 1 (MARCKSL1) is widely expressed in nervous tissue, where, like Jun N-terminal protein kinase (JNK), it is required for neural tube formation, though the mechanism is unknown. Here we show that MARCKSL1 is directly phosphorylated by JNK on C-terminal residues (S120, T148, and T183). This phosphorylation enables MARCKSL1 to bundle and stabilize F-actin, increase filopodium numbers and dynamics, and retard migration in neurons. Conversely, when MARCKSL1 phosphorylation is inhibited, actin mobility increases and filopodium formation is compromised whereas lamellipodium formation is enhanced, as is cell migration. We find that MARCKSL1 mRNA is upregulated in a broad range of cancer types and that MARCKSL1 protein is strongly induced in primary prostate carcinomas. Gene knockdown in prostate cancer cells or in neurons reveals a critical role for MARCKSL1 in migration that is dependent on the phosphorylation state; phosphomimetic MARCKSL1 (MARCKSL1(S120D,T148D,T183D)) inhibits whereas dephospho-MARCKSL1(S120A,T148A,T183A) induces migration. In summary, these data show that JNK phosphorylation of MARCKSL1 regulates actin homeostasis, filopodium and lamellipodium formation, and neuronal migration under physiological conditions and that, when ectopically expressed in prostate cancer cells, MARCKSL1 again determines cell movement.

  14. The N-terminal Part of Arabidopsis thaliana Starch Synthase 4 Determines the Localization and Activity of the Enzyme.

    Science.gov (United States)

    Raynaud, Sandy; Ragel, Paula; Rojas, Tomás; Mérida, Ángel

    2016-05-13

    Starch synthase 4 (SS4) plays a specific role in starch synthesis because it controls the number of starch granules synthesized in the chloroplast and is involved in the initiation of the starch granule. We showed previously that SS4 interacts with fibrillins 1 and is associated with plastoglobules, suborganelle compartments physically attached to the thylakoid membrane in chloroplasts. Both SS4 localization and its interaction with fibrillins 1 were mediated by the N-terminal part of SS4. Here we show that the coiled-coil region within the N-terminal portion of SS4 is involved in both processes. Elimination of this region prevents SS4 from binding to fibrillins 1 and alters SS4 localization in the chloroplast. We also show that SS4 forms dimers, which depends on a region located between the coiled-coil region and the glycosyltransferase domain of SS4. This region is highly conserved between all SS4 enzymes sequenced to date. We show that the dimerization seems to be necessary for the activity of the enzyme. Both dimerization and the functionality of the coiled-coil region are conserved among SS4 proteins from phylogenetically distant species, such as Arabidopsis and Brachypodium This finding suggests that the mechanism of action of SS4 is conserved among different plant species. PMID:26969163

  15. c-Jun N-terminal kinase is required for vitamin E succinate-induced apoptosis in human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Kun Wu; Yan Zhao; Gui-Chang Li; Wei-Ping Yu

    2004-01-01

    AIM: To investigate the roles of c-Jun N-terminal kinase (JNK)signaling pathway in vitamin E succinate-induced apoptosis in human gastric cancer SGC-7901 cells.METHODS: Human gastric cancer cell lines (SGC-7901)were treated with vitamin E succinate (VES) at 5, 10, 20 mg/L.Succinic acid and vitamin E were used as vehicle controls and condition medium only as an untreated (UT) control.Apoptosis was observed by 4′, 6-diamidine-2′-phenylindole dihydrochloride (DAPI) staining for morphological changes and by DNA fragmentation for biochemical alterations.Western blot analysis was applied to measure the expression ofJNK and phosphorylated JNK. After the cells were transiently transfected with dominant negative mutant of JNK (DNJNK) followed by treatment of VES, the expression of JNK and c-Jun protein was determined.RESULTS: The apoptotic changes were observed after VES treatment by DNA fragmentation. DNA ladder in the 20 mg/L VES group was more clearly seen than that in 10 mg/L VES group and was not detected following treatment of UT control, succinate and vitamin E. VES at 5, 10 and 20 mg/L increased the expression of p-JNK by 2.5-, 2.8- and 4.2-fold, respectively. VES induced the phosphorylation of JNK beginning at 1.5 h and produced a sustained increase for 24 h with the peak level at 12 h. Transient transfection of DN-JNK blocked VES-triggered apoptosis by 52%. DN-JNK significantly increased the level of JNK, while decreasing the expression of VES-induced c-Jun protein.CONCLUSION: VES-induced apoptosis in human gastric cancer SGC-7901 cells involves JNK signaling pathway via c-Jun and its downstream transcription factor.

  16. Modulating the activity of short arginine-tryptophan containing antibacterial peptides with N-terminal metallocenoyl groups

    Directory of Open Access Journals (Sweden)

    H. Bauke Albada

    2012-10-01

    Full Text Available A series of small synthetic arginine and tryptophan containing peptides was prepared and analyzed for their antibacterial activity. The effect of N-terminal substitution with metallocenoyl groups such as ferrocene (FcCO and ruthenocene (RcCO was investigated. Antibacterial activity in different media, growth inhibition, and killing kinetics of the most active peptides were determined. The toxicity of selected derivatives was determined against erythrocytes and three human cancer cell lines. It was shown that the replacement of an N-terminal arginine residue with a metallocenoyl moiety modulates the activity of WRWRW-peptides against Gram-positive and Gram-negative bacteria. MIC values of 2–6 µM for RcCO-W(RW2 and 1–11 µM for (RW3 were determined. Interestingly, W(RW2-peptides derivatized with ferrocene were significantly less active than those derivatized with ruthenocene which have similar structural but different electronic properties, suggesting a major influence of the latter. The high activities observed for the RcCO-W(RW2- and (RW3-peptides led to an investigation of the origin of activity of these peptides using several important activity-related parameters. Firstly, killing kinetics of the RcCO-W(RW2-peptide versus killing kinetics of the (RW3 derivative showed faster reduction of the colony forming units for the RcCO-W(RW2-peptide, although MIC values indicated higher activity for the (RW3-peptide. This was confirmed by growth inhibition studies. Secondly, hemolysis studies revealed that both peptides did not lead to significant destruction of erythrocytes, even up to 500 µg/mL for (RW3 and 250 µg/mL for RcCO-W(RW2. In addition, toxicity against three human cancer cell lines (HepG2, HT29, MCF7 showed that the (RW3-peptide had an IC50 value of ~140 µM and the RcW(RW2 one of ~90 µM, indicating a potentially interesting therapeutic window. Both the killing kinetics and growth inhibition studies presented in this work point to a

  17. Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor

    Directory of Open Access Journals (Sweden)

    Carolin Lübker

    2015-07-01

    Full Text Available Bacillus anthracis adenylyl cyclase toxin edema factor (EF is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH-oxidase, thus reducing production of reactive oxygen species (ROS used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut with Met to leucine (Leu substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils.

  18. c-Jun N-terminal kinases 3 (JNK3) from orange-spotted grouper, Epinephelus coioides, inhibiting the replication of Singapore grouper iridovirus (SGIV) and SGIV-induced apoptosis.

    Science.gov (United States)

    Guo, Minglan; Wei, Jingguang; Zhou, Yongcan; Qin, Qiwei

    2016-12-01

    C-Jun N-terminal kinases (JNKs), a subgroup of serine-threonine protein kinases that activated by phosphorylation, are involve in physiological and pathophysiological processes. JNK3 is one of JNK proteins involved in JNK3 signaling transduction. In the present study, two JNK3 isoforms, Ec-JNK3 X1 and Ec-JNK3 X2, were cloned from orange-spotted grouper, Epinephelus coioides. Both Ec-JNK3 X1 and Ec-JNK3 X2 were mainly expressed in liver, gill, skin, brain and muscle of juvenile grouper. The relative expression of Ec-JNK3 X2 mRNA was much higher in muscle and gill than that of Ec-JNK3 X1. Isoform-specific immune response to challenges was revealed by the expression profiles in vivo. Immunofluorescence staining indicated that JNK3 was localized in the cytoplasm of grouper spleen (GS) cells and shown immune response to SGIV infection in vitro. Over-expressing Ec-JNK3 X1 and/or Ec-JNK3 X2 inhibited the SGIV infection and replication and the SGIV-induced apoptosis. To achieve the antiviral and anti-apoptosis activities, JNK3 promoted the activation of genes ISRE and type I IFN in the antiviral IFN signaling pathway, and inhibited the activation of transcription factors NF-κB and p53 relating to apoptosis, respectively. Ec-JNK3 X2 showed stronger activities in antivirus and anti-apoptosis than that of Ec-JNK3 X1. Our results not only define the characterization of JNK3 but also reveal new immune functions and the molecular mechanisms of JNK3 on iridoviruses infection and the virus-induced apoptosis.

  19. The N-terminal domain allosterically regulates cleavage and activation of the epithelial sodium channel.

    Science.gov (United States)

    Kota, Pradeep; Buchner, Ginka; Chakraborty, Hirak; Dang, Yan L; He, Hong; Garcia, Guilherme J M; Kubelka, Jan; Gentzsch, Martina; Stutts, M Jackson; Dokholyan, Nikolay V

    2014-08-15

    The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr(370) in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation.

  20. The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel*

    Science.gov (United States)

    Kota, Pradeep; Buchner, Ginka; Chakraborty, Hirak; Dang, Yan L.; He, Hong; Garcia, Guilherme J. M.; Kubelka, Jan; Gentzsch, Martina; Stutts, M. Jackson; Dokholyan, Nikolay V.

    2014-01-01

    The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr370 in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation. PMID:24973914

  1. Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain

    Science.gov (United States)

    Hwang, I.; Harper, J. F.; Liang, F.; Sze, H.

    2000-01-01

    To investigate how calmodulin regulates a unique subfamily of Ca(2+) pumps found in plants, we examined the kinetic properties of isoform ACA2 identified in Arabidopsis. A recombinant ACA2 was expressed in a yeast K616 mutant deficient in two endogenous Ca(2+) pumps. Orthovanadate-sensitive (45)Ca(2+) transport into vesicles isolated from transformants demonstrated that ACA2 is a Ca(2+) pump. Ca(2+) pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta2-80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC(50) = 4 microM) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V(max) and increased the K(m) for Ca(2+) of the constitutively active ACA2-2 to values comparable to the full-length pump. The peptide also blocked the activity (IC(50) = 7 microM) of a Ca(2+) pump (AtECA1) belonging to a second family of Ca(2+) pumps. This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between aspartate-19 and arginine-36 (J.F. Harper, B. Hong, I. Hwang, H.Q. Guo, R. Stoddard, J.F. Huang, M.G. Palmgren, H. Sze inverted question mark1998 J Biol Chem 273: 1099-1106). These results support a model in which the pump is kept "unactivated" by an intramolecular interaction between an autoinhibitory sequence located between residues 20 and 44 and a site in the Ca(2+) pump core that is highly conserved between different Ca(2+) pump families. Results further support a model in which activation occurs as a result of Ca(2+)-induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.

  2. N-terminal guanidinylation of TIPP (Tyr-Tic-Phe-Phe) peptides results in major changes of the opioid activity profile

    OpenAIRE

    Weltrowska, Grazyna; Nguyen, Thi M.-D.; Chung, Nga N.; Wilkes, Brian C.; Schiller, Peter W.

    2013-01-01

    Derivatives of peptides of the TIPP (Tyr-Tic-Phe-Phe; Tic = 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid) family containing a guanidino (Guan) function in place of the N-terminal amino group were synthesized in an effort to improve their blood-brain barrier permeability. Unexpectedly, N-terminal amidination significantly altered the in vitro opioid activity profiles. Guan-analogues of TIPP-related δ opioid antagonists showed δ partial agonist or mixed δ partial agonist/μ partial agonist ...

  3. The N-terminal domain is a transcriptional activation domain required for Nanog to maintain ES cell self-renewal

    Institute of Scientific and Technical Information of China (English)

    GUO YunQian; ZHANG Juan; YE Li; CHEN Mo; YAO Dong; PAN GuangJin; ZHANG JieQiong; PEI DuanQing

    2009-01-01

    Nanog is a transcription factor identified by its ability to maintain the self-renewal of ES cells in the absence of leukemia inhibitory factor (LIF). Nanog protein contains an N-terminal domain (ND),a DNA-binding homeobox domain (HD) and a C-terminal domain (CD). We previously reported that the CD in Nanog is a transcriptional activation domain essential for the in vivo function of Nanog. Here we demonstrated that the ND in Nanog is also functionally important. Deletion of the ND reduces the transcriptional activity of Nanog on either artificial reporters or native Nanog promoters. This truncated Nanog is also less effective in regulating the endogenous Nanog target genes. Furthermore,the ND truncation disrupted the ability of Nanog to maintain ES cell self-renewal as well. We found that the ND Is not required for the nuclear localization of Nanog. These results suggest that the regulation of endogenous pluripotent genes such as oct3/4 and rex-1 is required for the in vivo function of Nanog.

  4. Generation and characterization of polyclonal antibodies specific to N-terminal extension of p85 isoform of ribosomal protein S6 kinase 1 (p85 S6K1

    Directory of Open Access Journals (Sweden)

    Savinska L. O.

    2015-08-01

    Full Text Available Aim. Generation of polyclonal antibodies specific to the ribosomal protein S6 kinase isoform – p85S6K1 and directed to the N-terminal (1–23 aa extension of p85S6K1. Methods. Animal immunization with synthetic (1–23 aa peptide, ELISA, Western blot, Immunoprecipitation, immunofluorescent analysis. Results. Polyclonal antibodies have been generated, which specifically recognize only p85 but not p70 isoform of S6K1 in western blot, immunoprecipitation and immunofluorescence analysis. Conclusions. The obtained antibodies can be recommended for studies on the p85S6K1 and other S6K1 isoforms possessing the N-terminal extension – the identification of binding protein partners, analysis of subcellular localization under different physiological conditions, elucidation of the signal transduction pathways involving different S6K1 isoforms.

  5. Salmonella induces SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling pathways in commercial and wild-type turkey leukocytes

    Science.gov (United States)

    Previous studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on d...

  6. Investigating the role of c-Jun N-terminal kinases in the proliferation of Werner syndrome fibroblasts using diaminopyridine inhibitors

    Directory of Open Access Journals (Sweden)

    Davis Terence

    2011-12-01

    Full Text Available Abstract Fibroblasts derived from the progeroid Werner syndrome show reduced replicative lifespan and a "stressed" morphology, both alleviated using the MAP kinase inhibitor SB203580. However, interpretation of these data is problematical because although SB203580 has the stress-activated kinases p38 and JNK1/2 as its preferred targets, it does show relatively low overall kinase selectivity. Several lines of data support a role for both p38 and JNK1/2 activation in the control of cellular proliferation and also the pathology of diseases of ageing, including type II diabetes, diseases to which Werner Syndrome individuals are prone, thus making the use of JNK inhibitors attractive as possible therapeutics. We have thus tested the effects of the widely used JNK inhibitor SP600125 on the proliferation and morphology of WS cells. In addition we synthesised and tested two recently described aminopyridine based inhibitors. SP600125 treatment resulted in the cessation of proliferation of WS cells and resulted in a senescent-like cellular phenotype that does not appear to be related to the inhibition of JNK1/2. In contrast, use of the more selective aminopyridine CMPD 6o at concentrations that fully inhibit JNK1/2 had a positive effect on cellular proliferation of immortalised WS cells, but no effect on the replicative lifespan of primary WS fibroblasts. In addition, CMPD 6o corrected the stressed WS cellular morphology. The aminopyridine CMPD 6r, however, had little effect on WS cells. CMDP 6o was also found to be a weak inhibitor of MK2, which may partially explain its effects on WS cells, since MK2 is known to be involved in regulating cellular morphology via HSP27 phosphorylation, and is thought to play a role in cell cycle arrest. These data suggest that total JNK1/2 activity does not play a substantial role in the proliferation control in WS cells.

  7. Chimeric RXFP1 and RXFP2 Receptors Highlight the Similar Mechanism of Activation Utilizing Their N-Terminal Low-Density Lipoprotein Class A Modules

    OpenAIRE

    Bruell, Shoni; Kong, Roy C. K.; Petrie, Emma J.; Hoare, Brad; John D Wade; Scott, Daniel J.; Gooley, Paul R.; Bathgate, Ross A. D.

    2013-01-01

    Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low-density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are...

  8. Chimeric RXFP1 and RXFP2 receptors highlight the similar mechanism of activation utilizing their N-terminal low density lipoprotein class A modules

    OpenAIRE

    RossBathgate; EmmaJunePetrie; JohnDWade

    2013-01-01

    Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are...

  9. Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

    DEFF Research Database (Denmark)

    Ngo, HT; Pham, Long; Kim, JW;

    2013-01-01

    Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray......, approximately 100 cellular proteins were identified as HCV core-interacting partners. Of these candidates, mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) was selected for further characterization. MAPKAPK3 is a serine/threonine protein kinase that is activated by stress and growth...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA...

  10. The N-terminal cellulose-binding domain of EGXA increases thermal stability of xylanase and changes its specific activities on different substrates

    Institute of Scientific and Technical Information of China (English)

    Ming Ding; Yigang Teng; Qiuyu Yin; Jie Zhao; Fukun Zhao

    2008-01-01

    A full-length EGXA enzyme from a mollusk, Ampullaria crossean, was cloned into pFastBac vector and then heterogeneously expressed in insect Tn5 cells. Its natural N-terminal signal peptide worked well in the insect Tn5 cells.The recombinant EGXA was a 63 kDa protein and had active endo-β-1,4-glucanase (EC 3.2.1.4) and endo-β-1,4-xylanase (EC 3.2.1.8). The specific activity of endo-β-1,4-xylanase was higher than in the EGX, which was purified from the stomach tissues of Ampullaria crossen. The N-terminal cellulosebinding domain of EGXA made it bind to cellulose and xylan more efficiently. This cellulose-binding domain also increased the thermal stability of this recombinant enzyme and decreased the recombinant EGXA's specific activities on p-nitrophenyi-β-D-cellobioside and sodium carboxymethyl cellulose.

  11. Mesenchymal stem cells promote liver regeneration and prolong survival in small-for-size liver grafts: involvement of C-Jun N-terminal kinase, cyclin D1, and NF-κB.

    Directory of Open Access Journals (Sweden)

    Weijie Wang

    Full Text Available BACKGROUND: The therapeutic potential of mesenchymal stem cells (MSCs has been highlighted recently for treatment of acute or chronic liver injury, by possibly differentiating into hepatocyte-like cells, reducing inflammation, and enhancing tissue repair. Despite recent progress, exact mechanisms of action are not clearly elucidated. In this study, we attempted to explore whether and how MSCs protected hepatocytes and stimulated allograft regeneration in small-for-size liver transplantation (SFSLT. METHODS: SFSLT model was established with a 30% partial liver transplantation (30PLT in rats. The differentiation potential and characteristics of bone marrow derived MSCs were explored in vitro. MSCs were infused transvenously immediately after graft implantation in therapy group. Expressions of apoptosis-, inflammatory-, anti-inflammatory-, and growth factor-related genes were measured by RT-PCR, activities of transcription factors AP-1 and NF-κB were analyzed by EMSA, and proliferative responses of the hepatic graft were evaluated by immunohistochemistry and western blot. RESULTS: MSCs were successfully induced into hepatocyte-like cells, osteoblasts and adipocytes in vitro. MSCs therapy could not only alleviate ischemia reperfusion injury and acute inflammation to promote liver regeneration, but also profoundly improve one week survival rate. It markedly up-regulated the mRNA expressions of HGF, Bcl-2, Bcl-XL, IL-6, IL-10, IP-10, and CXCR2, however, down-regulated TNF-α. Increased activities of AP-1 and NF-κB, as well as elevated expressions of p-c-Jun, cyclin D1, and proliferating cell nuclear antigen (PCNA, were also found in MSCs therapy group. CONCLUSION: These data suggest that MSCs therapy promotes hepatocyte proliferation and prolongs survival in SFSLT by reducing ischemia reperfusion injury and acute inflammation, and sustaining early increased expressions of c-Jun N-terminal Kinase, Cyclin D1, and NF-κB.

  12. c-Jun N-terminal kinase inhibitor favors transforming growth factor-β to antagonize hepatitis B virus X protein-induced cell growth promotion in hepatocellular carcinoma.

    Science.gov (United States)

    Wu, Yan-Hui; Ai, Xi; Liu, Fu-Yao; Liang, Hui-Fang; Zhang, Bi-Xiang; Chen, Xiao-Ping

    2016-02-01

    Transforming growth factor (TGF)-β induces cell growth arrest in well-differentiated hepatocellular carcinoma (HCC) while hepatitis B virus X protein (HBx) minimizes the tumor suppression of TGF-β signaling in early chronic hepatitis B. However, how to reverse the oncogenic effect of HBx and sustain the tumor-suppressive action of TGF-β has yet to be investigated. The present study examined the effect of TGF-β and a c-Jun N-terminal kinase (JNK) inhibitor on cell growth in HCC cells with forced expression of HBx. It was found that HBx promoted cell growth via activation of the JNK/pSMAD3L pathway and inhibition of the transforming growth factor-beta type I receptor (TβRI)/pSMAD3C pathway. pSMAD3L/SMAD4 and pSMAD3C/SMAD4 complexes antagonized each other to regulate c-Myc expression. In the absence of HBx, TGF-β induced cell growth arrest through activation of the TβRI/pSMAD3C pathway in well-differentiated HCC cells. In the presence of HBx, TGF-β had no effect on cell growth. JNK inhibitor SP600125 significantly reversed the oncogenic action of HBx and favored TGF-β to regain the ability to inhibit the cell growth in HBx-expressing well-differentiated HCC cells. In conclusion, targeting JNK signaling favors TGF-β to block HBx-induced cell growth promotion in well-differentiated HCC cells. As an adjunct to anti-viral therapy, the combination of TGF-β and inhibition of JNK signaling is a potential therapy for HBV-infected HCC.

  13. Protein kinase domain of twitchin has protein kinase activity and an autoinhibitory region.

    Science.gov (United States)

    Lei, J; Tang, X; Chambers, T C; Pohl, J; Benian, G M

    1994-08-19

    Twitchin is a 753-kDa polypeptide located in the muscle A-bands of the nematode, Caenorhabditis elegans. It consists of multiple copies of both fibronectin III and immunoglobulin C2 domains and, near the C terminus, a protein kinase domain with greatest homology to the catalytic domains of myosin light chain kinases. We have expressed and purified from Escherichia coli twitchin's protein kinase catalytic core and flanking sequences that do not include fibronectin III and immunoglobulin C2 domains. The protein was shown to phosphorylate a model substrate and to undergo autophosphorylation. The autophosphorylation occurs at a slow rate, attaining a maximum at 3 h with a stoichiometry of about 1.0 mol of phosphate/mol of protein, probably through an intramolecular mechanism. Sequence analysis of proteolytically derived phosphopeptides revealed that autophosphorylation occurred N-terminal to the catalytic core, predominantly at Thr-5910, with possible minor sites at Ser5912 and/or Ser-5913. This portion of twitchin (residues 5890-6268) was also phosphorylated in vitro by protein kinase C in the absence of calcium and phosphotidylserine, but not by cAMP-dependent protein kinase. By comparing the activities of three twitchin segments, the enzyme appears to be inhibited by the 60-amino acid residues lying just C-terminal to the kinase catalytic core. Thus, like a number of other protein kinases including myosin light chain kinases, the twitchin kinase appears to be autoregulated. PMID:8063727

  14. Neuroprotection by inhibiting the c-Jun N-terminal kinase pathway after cerebral ischemia occurs independently of interleukin-6 and keratinocyte-derived chemokine (KC/CXCL1 secretion

    Directory of Open Access Journals (Sweden)

    Benakis Corinne

    2012-04-01

    Full Text Available Abstract Background Cerebral ischemia is associated with the activation of glial cells, infiltration of leukocytes and an increase in inflammatory mediators in the ischemic brain and systemic circulation. How this inflammatory response influences lesion size and neurological outcome remains unclear. D-JNKI1, an inhibitor of the c-Jun N-terminal kinase pathway, is strongly neuroprotective in animal models of stroke. Intriguingly, the protection mediated by D-JNKI1 is high even with intravenous administration at very low doses with undetectable drug levels in the brain, pointing to a systemic mode of action, perhaps on inflammation. Findings We evaluated whether D-JNKI1, administered intravenously 3 h after the onset of middle cerebral artery occlusion (MCAO, modulates secretion of the inflammatory mediators interleukin-6 and keratinocyte-derived chemokine in the plasma and from the spleen and brain at several time points after MCAO. We found an early release of both mediators in the systemic circulation followed by an increase in the brain and went on to show a later systemic increase in vehicle-treated mice. Release of interleukin-6 and keratinocyte-derived chemokine from the spleen of mice with MCAO was not significantly different from sham mice. Interestingly, the secretion of these inflammatory mediators was not altered in the systemic circulation or brain after successful neuroprotection with D-JNKI1. Conclusions We demonstrate that neuroprotection with D-JNKI1 after experimental cerebral ischemia is independent of systemic and brain release of interleukin-6 and keratinocyte-derived chemokine. Furthermore, our findings suggest that the early systemic release of interleukin-6 and keratinocyte-derived chemokine may not necessarily predict an unfavorable outcome in this model.

  15. C-Jun N-terminal kinase signal pathway and C-Jun N-terminal kinase inhibitor SP600125 in amygdala kindled rats%c-Jun氨基末端激酶信号通路及其抑制剂SP600125在大鼠杏仁核电刺激癫痫模型中的作用

    Institute of Scientific and Technical Information of China (English)

    吴俊; 陈旭; 舒凯; 肖铮铮; 雷霆; 李龄

    2012-01-01

    Objective By injecting SP600125 into ventricle of amygdale kindled rats,to observe the pathological changes of the hippocampus and the change of C-Jun N-terminal kinase (JNK) phosphorylation,and discuss the action mechanism of SP600125.Methods Forty rats were randomly divided into 4 groups (n =10 each):blank group,kindling group,SP600125 group,DMSO group.Whole-cell extracts of tissues were obtained from the right hippocampus,and Western blotting was used to detect the changes of JNK and phosphorylation of JNK.Pathological changes of the hippocampus and amygdla were observed by GFAP stain and Nissl stain.Results The level of JNK phosphorylation in the hippocampus was significantly higher in the kindling group (0.48 ± 0.04 ) than the blank group (0.38 ± 0.04 ) and the SP600125 group (0.37±0.03).Nissl stain positive cells in the hippocampus of the SP600125 group were significantly more than those in the the DMSO group (20.10 ±5.11 ).The expression of GFAP in the hippocampus of kindling group (65.45 ±4.53 ) and DMSO group (67.18 ± 3.52) was significantly stronger than that in the blank group (40.37 ± 3.82) and the SP600125 group (43.51 ± 1.83).Conclusion The role of repeated activation of JNK can be related to the hippocampal sclerosis in these rats.SP600125 had a protective effect on neurons during the kindling procedure.%目的 通过对杏仁核电刺激癫痫模型大鼠脑室内注射c-Jun氨基末端激酶(JNK)特异性抑制剂SP600125,观察海马区的病理变化和JNK水平的变化,探讨SP600125的作用.方法 将40只Wistar大鼠随机分为4组:空白组、点燃组、加药组和加药对照组各10只,10次癫痫发作后灌注取脑,Western blot法检测JNK的表达变化,进行尼氏和胶原纤维酸性蛋白(GFAP)染色,各组间进行比较.结果 Western blot显示点燃组海马区的JNK磷酸化水平(0.48±0.04)较空白组(0.38±0.04)和加药组(0.37±0.03)显著增高(P<0.05),总JNK水平各组之间差异无统计学意义(P>0

  16. Identification of quercitrin as an inhibitor of the p90 S6 ribosomal kinase (RSK): structure of its complex with the N-terminal domain of RSK2 at 1.8 Å resolution

    Energy Technology Data Exchange (ETDEWEB)

    Derewenda, Urszula; Artamonov, Mykhaylo; Szukalska, Gabriela; Utepbergenov, Darkhan; Olekhnovich, Natalya [University of Virginia, Charlottesville, VA 22908-0736 (United States); Parikh, Hardik I.; Kellogg, Glen E. [Virginia Commonwealth University, Richmond, VA 23298-0540 (United States); Somlyo, Avril V.; Derewenda, Zygmunt S., E-mail: zsd4n@virginia.edu [University of Virginia, Charlottesville, VA 22908-0736 (United States)

    2013-02-01

    The crystal structure of quercitrin, a naturally occurring flavonol glycoside, has been determined in a complex with the N-terminal kinase domain of murine RSK2. The structure revealed that quercitrin inhibits the RSK2 kinase in the same fashion as another known inhibitor, SL0101. Members of the RSK family of kinases constitute attractive targets for drug design, but a lack of structural information regarding the mechanism of selective inhibitors impedes progress in this field. The crystal structure of the N-terminal kinase domain (residues 45–346) of mouse RSK2, or RSK2{sup NTKD}, has recently been described in complex with one of only two known selective inhibitors, a rare naturally occurring flavonol glycoside, kaempferol 3-O-(3′′,4′′-di-O-acetyl-α-l-rhamnopyranoside), known as SL0101. Based on this structure, it was hypothesized that quercitrin (quercetin 3-O-α-l-rhamnopyranoside), a related but ubiquitous and inexpensive compound, might also act as an RSK inhibitor. Here, it is demonstrated that quercitrin binds to RSK2{sup NTKD} with a dissociation constant (K{sub d}) of 5.8 µM as determined by isothermal titration calorimetry, and a crystal structure of the binary complex at 1.8 Å resolution is reported. The crystal structure reveals a very similar mode of binding to that recently reported for SL0101. Closer inspection shows a number of small but significant differences that explain the slightly higher K{sub d} for quercitrin compared with SL0101. It is also shown that quercitrin can effectively substitute for SL0101 in a biological assay, in which it significantly suppresses the contractile force in rabbit pulmonary artery smooth muscle in response to Ca{sup 2+}.

  17. Molecular basis for activation of G protein-coupled receptor kinases

    Energy Technology Data Exchange (ETDEWEB)

    Boguth, Cassandra A.; Singh, Puja; Huang, Chih-chin; Tesmer, John J.G. (Michigan)

    2012-03-16

    G protein-coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N-terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N-terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.

  18. Knockout of the c-Jun N-terminal Kinase 2 aggravates the development of mild chronic dextran sulfate sodium colitis independently of expression of intestinal cytokines TNFα, TGFB1, and IL-6

    Directory of Open Access Journals (Sweden)

    Kersting S

    2013-02-01

    Full Text Available Sabine Kersting,1 Kirstin Reinecke,2 Christoph Hilgert,1 Monika S Janot,1 Elisabeth Haarmann,1 Martin Albrecht,1 Annette M Müller,3 Thomas Herdegen,2 Ulrich Mittelkötter,1 Waldemar Uhl,1 Ansgar M Chromik11Department of General and Visceral Surgery, St Josef Hospital, Ruhr-University of Bochum, Bochum, Germany; 2Institute of Experimental and Clinical Pharmacology, University Hospital of Schleswig-Holstein, Campus Kiel, Germany; 3Department of Pediatric Pathology, Rheinische Friedrich-Wilhems-University of Bonn, Bonn, GermanyIntroduction: The c-Jun N-terminal kinases (JNKs are involved in signal transduction of inflammatory bowel diseases. The aim of this study was to examine the function of JNKs by using a low-dose dextran sulfate sodium (DSS model in JNK1 knockout mice (Mapk8–/–, JNK2 knockout mice (Mapk9–/–, and wild-type controls (WT1, WT2.Methods: The animals were evaluated daily using a disease activity index. After 30 days, the intestine was evaluated histologically with a crypt damage score. CD4+ and CD8+ cells were quantified using immunofluorescence. Analysis of tumor necrosis factor-a (TNFα, interleukin-6 (IL-6, and transforming growth factor ß1 (TGFB1 expression was carried out using LightCycler® real-time polymerase chain reaction.Results: Cyclic administration of low-dose DSS (1% was not able to induce features of chronic colitis in Mapk8–/– WT2 mice. By contrast, DSS administration significantly increased the disease activity index in WT1 and Mapk9–/– mice. In Mapk9–/– mice, the crypt damage score and the number of CD4+ and CD8+ cells as features of chronic colitis/inflammation were also significantly elevated. Expression of TNFα, IL-6, and TGFB1 was not altered by the JNK knockout.Conclusion: Administering DSS at a defined low concentration that is unable to induce colitis in WT animals leads to clinically and histologically detectable chronic colitis in Mapk9–/– mice. The reason for this disease

  19. Involvement of mitogen-activated protein kinase pathways in N-methyl-D-aspartate-induced excitotoxicity

    Institute of Scientific and Technical Information of China (English)

    Xiaorong Yang; Ping Sun; Huaping Qin; Rui Wang; Ye Wang; Ruihong Shi; Xin Zhao; Ce Zhang

    2011-01-01

    Previous studies have shown that mitogen-activated protein kinase (MAPK) signaling pathways are involved in N-methyl-D-aspartate (NMDA)-mediated excitotoxicity. However, a systematic observation or analysis of the role of these various MAPK pathways in excitotoxicity processes does not exist. The present study further evaluated the role and contribution of three MAPK pathways extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK in an NMDA-mediated excitotoxicity model using MAPK-specific inhibitor. Results demonstrated that c-Jun N-terminal kinase inhibitor SP600125 and/or p38 MAPK inhibitor SB203580 inhibited NMDA-induced reduction in cell viability, as well as reduced NMDA-induced lactate dehydrogenase leakage and reactive oxygen species production. However, PD98059, an inhibitor of extracellular signal-regulated kinase, did not influence this model. Results demonstrated an involvement of c-Jun N-terminal kinase and p38 MAPK, but not extracellular signal-regulated kinase, in NMDA-mediated excitotoxicity in cortical neurons.

  20. Streptococcus pneumoniae induced c-Jun-N-terminal kinase- and AP-1 -dependent IL-8 release by lung epithelial BEAS-2B cells

    Directory of Open Access Journals (Sweden)

    Rosseau Simone

    2006-07-01

    Full Text Available Abstract Background Although pneumococcal pneumonia is one of the most common causes of death due to infectious diseases, little is known about pneumococci-lung cell interaction. Herein we tested the hypothesis that pneumococci activated pulmonary epithelial cell cytokine release by c-Jun-NH2-terminal kinase (JNK Methods Human bronchial epithelial cells (BEAS-2B or epithelial HEK293 cells were infected with S. pneumoniae R6x and cytokine induction was measured by RT-PCR, ELISA and Bioplex assay. JNK-phosphorylation was detected by Western blot and nuclear signaling was assessed by electrophoretic mobility shift assay (EMSA and chromatin immunoprecipitation (ChIP. JNK was modulated by the small molecule inhibitor SP600125 and AP1 by transfection of a dominant negative mutant. Results S. pneumoniae induced the release of distinct CC and CXC, as well as Th1 and Th2 cytokines and growth factors by human lung epithelial cell line BEAS-2B. Furthermore, pneumococci infection resulted in JNK phosphorylation in BEAS-2B cells. Inhibition of JNK by small molecule inhibitor SP600125 reduced pneumococci-induced IL-8 mRNA expression and release of IL-8 and IL-6. One regulator of the il8 promoter is JNK-phosphorylated activator protein 1 (AP-1. We showed that S. pneumoniae time-dependently induced DNA binding of AP-1 and its phosphorylated subunit c-Jun with a maximum at 3 to 5 h after infection. Recruitment of Ser63/73-phosphorylated c-Jun and RNA polymerase II to the endogenous il8 promoter was found 2 h after S. pneumoniae infection by chromatin immunoprecipitation. AP-1 repressor A-Fos reduced IL-8 release by TLR2-overexpressing HEK293 cells induced by pneumococci but not by TNFα. Antisense-constructs targeting the AP-1 subunits Fra1 and Fra2 had no inhibitory effect on pneumococci-induced IL-8 release. Conclusion S. pneumoniae-induced IL-8 expression by human epithelial BEAS-2B cells depended on activation of JNK and recruitment of phosphorylated c

  1. Sepp1UF forms are N-terminal selenoprotein P truncations that have peroxidase activity when coupled with thioredoxin reductase-1

    OpenAIRE

    Kurokawa, Suguru; Eriksson, Sofi; Rose, Kristie L.; Wu, Sen; Motley, Amy K.; Hill, Salisha; Winfrey, Virginia P.; McDonald, W. Hayes; Capecchi, Mario R.; Atkins, John F; Arnér, Elias S. J.; Hill, Kristina E.; Raymond F Burk

    2014-01-01

    Mouse selenoprotein P (Sepp1) consists of an N-terminal domain (residues 1-239) that contains 1 selenocysteine (U) as residue 40 in a proposed redox-active motif (-UYLC-) and a C-terminal domain (residues 240-361) that contains 9 selenocysteines. Sepp1 transports selenium from the liver to other tissues by receptor-mediated endocytosis. It also reduces oxidative stress in vivo by an unknown mechanism. A previously uncharacterized plasma form of Sepp1 is filtered in the glomerulus and taken up...

  2. N-terminal guanidinylation of TIPP (Tyr-Tic-Phe-Phe) peptides results in major changes of the opioid activity profile.

    Science.gov (United States)

    Weltrowska, Grazyna; Nguyen, Thi M-D; Chung, Nga N; Wilkes, Brian C; Schiller, Peter W

    2013-09-15

    Derivatives of peptides of the TIPP (Tyr-Tic-Phe-Phe; Tic=1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) family containing a guanidino (Guan) function in place of the N-terminal amino group were synthesized in an effort to improve their blood-brain barrier permeability. Unexpectedly, N-terminal amidination significantly altered the in vitro opioid activity profiles. Guan-analogues of TIPP-related δ opioid antagonists showed δ partial agonist or mixed δ partial agonist/μ partial agonist activity. Guanidinylation of the mixed μ agonist/δ antagonists H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) and H-Dmt-TicΨ[CH2NH]Phe-Phe-NH2 (DIPP-NH2[Ψ]) converted them to mixed μ agonist/δ agonists. A docking study revealed distinct positioning of DIPP-NH2 and Guan-DIPP-NH2 in the δ receptor binding site. Lys(3)-analogues of DIPP-NH2 and DIPP-NH2[Ψ] (guanidinylated or non-guanidinylated) turned out to be mixed μ/κ agonists with δ antagonist-, δ partial agonist- or δ full agonist activity. Compounds with some of the observed mixed opioid activity profiles have therapeutic potential as analgesics with reduced side effects or for treatment of cocaine addiction. PMID:23932788

  3. The N-terminal cysteine-rich domain of tobacco class I chitinase is essential for chitin binding but not for catalytic or antifungal activity.

    Science.gov (United States)

    Iseli, B; Boller, T; Neuhaus, J M

    1993-09-01

    The vacuolar chitinases of class I possess an N-terminal cysteine-rich domain homologous to hevein and chitin-binding lectins such as wheat germ agglutinin and Urtica dioica lectin. To investigate the significance of this domain for the biochemical and functional characteristics of chitinase, chimeric genes encoding the basic chitinase A of tobacco (Nicotiana tabacum) with and without this domain were constructed and constitutively expressed in transgenic Nicotiana sylvestris. The chitinases were subsequently isolated and purified to homogeneity from the transgenic plants. Chromatography on colloidal chitin revealed that only the form with the N-terminal domain, and not the one without it, had chitin-binding properties, demonstrating directly that the domain is a chitin-binding domain (CBD). Under standard assay conditions with radioactive colloidal chitin, both forms of chitinase had approximately the same catalytic activity. However, kinetic analysis demonstrated that the enzyme without CBD had a considerably lower apparent affinity for its substrate. The pH and temperature optima of the two chitinases were similar, but the form with the CBD had an approximately 3-fold higher activation energy and retained a higher activity at low pH values. Both chitinases were capable of inhibiting growth of Trichoderma viride, although the form with the CBD was about three times more effective than the one without it. Thus, the CBD is not necessary for catalytic or antifungal activity of chitinase. PMID:8208848

  4. Characterization of an invertase with pH tolerance and truncation of its N-terminal to shift optimum activity toward neutral pH.

    Directory of Open Access Journals (Sweden)

    Liqin Du

    Full Text Available Most invertases identified to date have optimal activity at acidic pH, and are intolerant to neutral or alkaline environments. Here, an acid invertase named uninv2 is described. Uninv2 contained 586 amino acids, with a 100 amino acids N-terminal domain, a catalytic domain and a C-terminal domain. With sucrose as the substrate, uninv2 activity was optimal at pH 4.5 and at 45°C. Removal of N-terminal domain of uninv2 has shifted the optimum pH to 6.0 while retaining its optimum temperaure at 45°C. Both uninv2 and the truncated enzyme retained highly stable at neutral pH at 37°C, and they were stable at their optimum pH at 4°C for as long as 30 days. These characteristics make them far superior to invertase from Saccharomyces cerevisiae, which is mostly used as industrial enzyme.

  5. Hydrokinetic activity of secretion and secretin analogues, modified in the N-terminal sequence, and of vasoactive intestinal peptide in the dog pancreas.

    Science.gov (United States)

    Lehnert, P; Forell, M M; Jaeger, E; Moroder, L; Wünsch, E

    1981-01-01

    In the dog pancreas in vivo, the biological activity of secretin and vasoactive intestinal peptide was compared to that of secretin analogues modified in their N-terminal hexapeptide and to X-secretion (alpha, beta-Asp3-secretin) and Y-secretin (a conversion product of X-secretin consisting of about 15% secretin and 85% beta-Asp3-secretin). Replacement of Asp3 by glutamic acid reduced secretin activity markedly. Replacement by neutral amino acids abolished the activity nearly completely. alpha, beta-Asp3-secretin and beta-Asp3-secretin appeared to be ineffective. The results indicate that the free beta-carboxy group of the side chain of the Asp3 residue of the secretin molecule is of decisive importance for hydrokinetic action. PMID:7274610

  6. The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation*

    Science.gov (United States)

    Kong, Roy C. K.; Petrie, Emma J.; Mohanty, Biswaranjan; Ling, Jason; Lee, Jeremy C. Y.; Gooley, Paul R.; Bathgate, Ross A. D.

    2013-01-01

    The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true “ligand” of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation. PMID:23926099

  7. The relaxin receptor (RXFP1) utilizes hydrophobic moieties on a signaling surface of its N-terminal low density lipoprotein class A module to mediate receptor activation.

    Science.gov (United States)

    Kong, Roy C K; Petrie, Emma J; Mohanty, Biswaranjan; Ling, Jason; Lee, Jeremy C Y; Gooley, Paul R; Bathgate, Ross A D

    2013-09-27

    The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true "ligand" of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation. PMID:23926099

  8. The N-terminal hybrid binding domain of RNase HI from Thermotoga maritima is important for substrate binding and Mg2+-dependent activity.

    Science.gov (United States)

    Jongruja, Nujarin; You, Dong-Ju; Kanaya, Eiko; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

    2010-11-01

    Thermotoga maritima ribonuclease H (RNase H) I (Tma-RNase HI) contains a hybrid binding domain (HBD) at the N-terminal region. To analyze the role of this HBD, Tma-RNase HI, Tma-W22A with the single mutation at the HBD, the C-terminal RNase H domain (Tma-CD) and the N-terminal domain containing the HBD (Tma-ND) were overproduced in Escherichia coli, purified and biochemically characterized. Tma-RNase HI prefers Mg(2+) to Mn(2+) for activity, and specifically loses most of the Mg(2+)-dependent activity on removal of the HBD and 87% of it by the mutation at the HBD. Tma-CD lost the ability to suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that the HBD is responsible for in vivo RNase H activity. The cleavage-site specificities of Tma-RNase HI are not significantly changed on removal of the HBD, regardless of the metal cofactor. Binding analyses of the proteins to the substrate using surface plasmon resonance indicate that the binding affinity of Tma-RNase HI is greatly reduced on removal of the HBD or the mutation. These results indicate that there is a correlation between Mg(2+)-dependent activity and substrate binding affinity. Tma-CD was as stable as Tma-RNase HI, indicating that the HBD is not important for stability. The HBD of Tma-RNase HI is important not only for substrate binding, but also for Mg(2+)-dependent activity, probably because the HBD affects the interaction between the substrate and enzyme at the active site, such that the scissile phosphate group of the substrate and the Mg(2+) ion are arranged ideally.

  9. Oncoprotein protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Karin, Michael (San Diego, CA); Hibi, Masahiko (San Diego, CA); Lin, Anning (La Jolla, CA); Davis, Roger (Princeton, MA); Derijard, Benoit (Shrewsbury, MA)

    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.

  10. Role of mitogen- activated protein kinase in myocardial hypertrophy%丝裂原活化蛋白激酶信号途径在心肌肥厚中的作用进展

    Institute of Scientific and Technical Information of China (English)

    黄朝阳; 朱建华

    2005-01-01

    Myocardial hypertrophy is an independent risk factor for cardiac events. Mitogen-activated protein kinases(MAPK), including extracellular signal-regulated kinases, C-jun N-terminal kinases and P38-MAPK, are the common intracellular pathway of transducing hypertrophic signs. All three MAPK subfamilies play an important role in development of myocardial hypertrophy.

  11. Towards the N-terminal acetylome

    DEFF Research Database (Denmark)

    Zhang, Xumin; Højrup, Peter

    2013-01-01

    Protein N-terminal acetylation (N(α)-acetylation) is observed widely from prokaryotes to eukaryotes. It gains increased importance in biological field, due to its multiple roles in many aspects of the protein life, such as assembly, stability, activity, and location. Today, mass spectrometry (MS...

  12. Increase of RhoB in {gamma}-radiation-induced apoptosis is regulated by c-Jun N-terminal kinase in Jurkat T cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Chun-Ho [Laboratory of Cytogenetics and Tissue Regeneration, KIRAMS, Seoul 139-706 (Korea, Republic of); Won, Misun; Choi, Chung-Hae; Ahn, Jiwon; Kim, Bo-Kyung [Genome Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Song, Kyung-Bin [Department of Food Science and Technology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Kang, Chang-Mo, E-mail: kangcm@kcch.re.kr [Laboratory of Cytogenetics and Tissue Regeneration, KIRAMS, Seoul 139-706 (Korea, Republic of); Chung, Kyung-Sook, E-mail: kschung@kribb.re.kr [Genome Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of)

    2010-01-08

    The Ras-related small GTP-binding protein RhoB is known to be a pro-apoptotic protein and immediate-early inducible by genotoxic stresses. In addition, JNK activation is known to function in {gamma}-radiation-induced apoptosis. However, it is unclear how JNK activation and {gamma}-radiation-dependent RhoB induction are related. Here we verified the relationship between JNK activation and RhoB induction. RhoB induction by {gamma}-radiation occurred at the transcriptional level and transcriptional activation of RhoB was concomitant with an increase in RhoB protein. {gamma}-Radiation-induced RhoB expression was markedly attenuated by pretreatment with a JNK-specific inhibitor, SP600125, but not by a p38 MAPK inhibitor, SB203580. Inhibition of JNK caused a decrease in early apoptotic cell death that correlated with RhoB expression. However, PI3K inhibition had no significant effects, indicating that the AKT survival pathway was not involved. The siRNA knockdown of JNK resulted in a decrease in RhoB expression and the siRNA knockdown of RhoB restored cell growth even in the {gamma}-irradiated cells. These results suggest that RhoB regulation involves the JNK pathway and contributes to the early apoptotic response of Jurkat T cells to {gamma}-radiation.

  13. Identifying the activation motif in the N-terminal of rainbow trout and zebrafish melanocortin-2 receptor accessory protein 1 (MRAP1) orthologs.

    Science.gov (United States)

    Dores, Robert M; Liang, Liang; Hollmann, Rebecca E; Sandhu, Navdeep; Vijayan, Mathilakath M

    2016-08-01

    The activation of mammalian melanocortin-2 receptor (MC2R) orthologs is dependent on a four-amino acid activation motif (LDYL/I) located in the N-terminal of mammalian MRAP1 (melanocortin-2 receptor accessory protein). Previous alanine substitution analysis had shown that the Y residue in this motif appears to be the most important for mediating the activation of mammalian MC2R orthologs. Similar, but not identical amino acid motifs were detected in rainbow trout MRAP1 (YDYL) and zebrafish MRAP1 (YDYV). To determine the importance of these residues in the putative activation motifs, rainbow trout and zebrafish MRAP1 orthologs were individually co-expressed in CHO cells with rainbow trout MC2R, and the activation of this receptor with either the wild-type MRAP1 ortholog or alanine-substituted analogs of the two teleost MRAP1s was analyzed. Alanine substitutions at all four amino acid positions in rainbow trout MRAP1 blocked activation of the rainbow trout MC2R. Single alanine substitutions of the D and Y residues in rainbow trout and zebrafish MRAP1 indicate that these two residues play a significant role in the activation of rainbow trout MC2R. These observations indicate that there are subtle differences in the way that teleost and mammalian MRAPs are involved in the activation of their corresponding MC2R orthologs. PMID:26752246

  14. 4-hydroxy-2, 3-nonenal activates activator protein-1 and mitogen-activated protein kinases in rat pancreatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Kazuhiro Kikuta; Atsushi Masamune; Masahiro Satoh; Noriaki Suzuki; Tooru Shimosegawa

    2004-01-01

    AIM: Activated pancreatic stellate cells (PSCs) are implicated in the pathogenesis of pancreatic inflammation and fibrosis,where oxidative stress is thought to play a key role. 4-hydroxy2,3-nonenal (HNE) is generated endogenously during the process of lipid peroxidation, and has been accepted as a mediator of oxidative stress. The aim of this study was to clarify the effects of HNE on the activation of signal transduction pathways and cellular functions in PSCs.METHODS: PSCs were isolated from the pancreas of male Wistar rats after perfusion with collagenase P, and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. PSCs were treated with physiologically relevant and non-cytotoxic concentrations (up to 5 μmol/L)of HNE. Activation of transcription factors was examined by electrophoretic mobility shift assay and luciferase assay.Activation of mitogen-activated protein (MAP) kinases was assessed by Western blotting using anti-phosphospecific antibodies. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine. Production of type Ⅰ collagen and monocyte chemoattractant protein-1was determined by enzyme-linked immunosorbent assay.The effect of HNE on the transformation of freshly isolated PSCs in culture was also assessed.RESULTS: HNE activated activator protein-1, but not nuclear factor κB. In addition, HNE activated three classes of MAP kinases: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAP kinase. HNE increased type Ⅰ collagen production through the activation of p38 MAP kinase and c-Jun N-terminal kinase. HNE did not alter the proliferation,or monocyte chemoattractant protein-1 production. HNE did not initiate the transformation of freshly isolated PSCs to myofibroblast-like phenotype.CONCLUSION: Specific activation of these signal transduction pathways and altered cell functions such as collagen production by HNE may play a role in the pathogenesis of pancreatic

  15. c-jun N-terminal kinase is involved in AUUUA-mediated interleukin-3 mRNA turnover in mast cells.

    OpenAIRE

    Ming, X F; Kaiser, M.; Moroni, C

    1998-01-01

    Whereas signalling pathways involved in transcriptional control have been studied extensively, the pathways regulating mRNA turnover remain poorly understood. We are interested in the role of mRNA stability in cell activation and oncogenesis using PB-3c mast cells as a model system. In these cells the short-lived interleukin-3 (IL-3) mRNA is stabilized by ionomycin treatment and following oncogenesis. To identify the signalling pathways involved in these mechanisms, we analysed the effect of ...

  16. Structure-activity relationships of the antimicrobial peptide arasin 1 - and mode of action studies of the N-terminal, proline-rich region.

    Directory of Open Access Journals (Sweden)

    Victoria S Paulsen

    Full Text Available Arasin 1 is a 37 amino acid long proline-rich antimicrobial peptide isolated from the spider crab, Hyas araneus. In this work the active region of arasin 1 was identified through structure-activity studies using different peptide fragments derived from the arasin 1 sequence. The pharmacophore was found to be located in the proline/arginine-rich NH(2 terminus of the peptide and the fragment arasin 1(1-23 was almost equally active to the full length peptide. Arasin 1 and its active fragment arasin 1(1-23 were shown to be non-toxic to human red blood cells and arasin 1(1-23 was able to bind chitin, a component of fungal cell walls and the crustacean shell. The mode of action of the fully active N-terminal arasin 1(1-23 was explored through killing kinetic and membrane permeabilization studies. At the minimal inhibitory concentration (MIC, arasin 1(1-23 was not bactericidal and had no membrane disruptive effect. In contrast, at concentrations of 5×MIC and above it was bactericidal and interfered with membrane integrity. We conclude that arasin 1(1-23 has a different mode of action than lytic peptides, like cecropin P1. Thus, we suggest a dual mode of action for arasin 1(1-23 involving membrane disruption at peptide concentrations above MIC, and an alternative mechanism of action, possibly involving intracellular targets, at MIC.

  17. Mitogen-Activated Protein Kinases and Hypoxic/Ischemic Nephropathy

    Directory of Open Access Journals (Sweden)

    Fengbao Luo

    2016-08-01

    Full Text Available Tissue hypoxia/ischemia is a pathological feature of many human disorders including stroke, myocardial infarction, hypoxic/ischemic nephropathy, as well as cancer. In the kidney, the combination of limited oxygen supply to the tissues and high oxygen demand is considered the main reason for the susceptibility of the kidney to hypoxic/ischemic injury. In recent years, increasing evidence has indicated that a reduction in renal oxygen tension/blood supply plays an important role in acute kidney injury, chronic kidney disease, and renal tumorigenesis. However, the underlying signaling mechanisms, whereby hypoxia alters cellular behaviors, remain poorly understood. Mitogen-activated protein kinases (MAPKs are key signal-transducing enzymes activated by a wide range of extracellular stimuli, including hypoxia/ischemia. There are four major family members of MAPKs: the extracellular signal-regulated kinases-1 and -2 (ERK1/2, the c-Jun N-terminal kinases (JNK, p38 MAPKs, and extracellular signal-regulated kinase-5 (ERK5/BMK1. Recent studies, including ours, suggest that these MAPKs are differentially involved in renal responses to hypoxic/ischemic stress. This review will discuss their changes in hypoxic/ischemic pathophysiology with acute kidney injury, chronic kidney diseases and renal carcinoma.

  18. N-terminal amino acids of bovine alpha interferons are relevant for the neutralization of their antiviral activity

    Directory of Open Access Journals (Sweden)

    Barreto Filho J.B.

    2001-01-01

    Full Text Available The structure-function relationship of interferons (IFNs has been studied by epitope mapping. Epitopes of bovine IFNs, however, are practically unknown, despite their importance in virus infections and in the maternal recognition of pregnancy. It has been shown that recombinant bovine (rBoIFN-alphaC and rBoIFN-alpha1 differ only in 12 amino acids and that the F12 monoclonal antibody (mAb binds to a linear sequence of residues 10 to 34. We show here that the antiviral activities of these two IFNs were neutralized by the F12 mAb to different extents using two tests. In residual activity tests the antiviral activity dropped by more than 99% with rBoIFN-alphaC and by 84% with rBoIFN-alpha1. In checkerboard antibody titrations, the F12 mAb titer was 12,000 with rBoIFN-alphaC and only 600 with rBoIFN-alpha1. Since these IFNs differ in their amino acid sequence at positions 11, 16 and 19 of the amino terminus, only these amino acids could account for the different neutralization titers, and they should participate in antibody binding. According to the three-dimensional structure described for human and murine IFNs, these amino acids are located in the alpha helix A; amino acids 16 and 19 of the bovine IFNs would be expected to be exposed and could bind to the antibody directly. The amino acid at position 11 forms a hydrogen bond in human IFNs-alpha and it is possible that, in bovine IFNs-alpha, the F12 mAb, binding near position 11, would disturb this hydrogen bond, resulting in the difference in the extent of neutralization observed.

  19. N-terminal domain of Bothrops asper Myotoxin II Enhances the Activity of Endothelin Converting Enzyme-1 and Neprilysin

    Science.gov (United States)

    Smith, A. Ian; Rajapakse, Niwanthi W.; Kleifeld, Oded; Lomonte, Bruno; Sikanyika, Nkumbu L.; Spicer, Alexander J.; Hodgson, Wayne C.; Conroy, Paul J.; Small, David H.; Kaye, David M.; Parkington, Helena C.; Whisstock, James C.; Kuruppu, Sanjaya

    2016-01-01

    Neprilysin (NEP) and endothelin converting enzyme-1 (ECE-1) are two enzymes that degrade amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49-P1-20, from the venom of Bothrops asper which directly enhances the activity of both ECE-1 and NEP. This is evidenced by a 2- and 5-fold increase in the Vmax of ECE-1 and NEP respectively. The K49-P1-20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE-1 and NEP was 1.92 ± 0.07 and 1.33 ± 0.12 μM respectively. Using BLITZ biolayer interferometry we have shown that K49-P1-20 interacts directly with each enzyme. Intrinsic fluorescence of the enzymes change in the presence of K49-P1-20 suggesting a change in conformation. ECE-1 mediated reduction in the level of endogenous soluble amyloid beta 42 in cerebrospinal fluid is significantly higher in the presence of K49-P1-20 (31 ± 4% of initial) compared with enzyme alone (11 ± 5% of initial; N = 8, P = 0.005, unpaired t-test). K49-P1-20 could be an excellent research tool to study mechanism(s) of enzyme stimulation, and a potential novel drug lead in the fight against Alzheimer’s disease. PMID:26931059

  20. Regulation of mitogen-activated protein kinase pathways by the plasma membrane Na+/H+ exchanger, NHE1

    DEFF Research Database (Denmark)

    Pedersen, Stine Helene Falsig; Darborg, Barbara Vasek; Rentsch, Maria Louise;

    2006-01-01

    The mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, play a major role in the regulation of pivotal cellular processes such as cell death/survival balance, cell cycle progression, and cell migration. MAPK...... activity is regulated by a three-tiered phosphorelay system, which is in turn regulated by a complex network of signaling events and scaffolding proteins. The ubiquitous plasma membrane Na(+)/H(+) exchanger NHE1 is activated by, and implicated in, the physiological/pathophysiological responses to many of...

  1. Chaperone-like activities of different molecular forms of beta-casein. Importance of polarity of N-terminal hydrophilic domain.

    Science.gov (United States)

    Yousefi, Reza; Shchutskaya, Yulia Y; Zimny, Jaroslaw; Gaudin, Jean-Charles; Moosavi-Movahedi, Ali A; Muronetz, Vladimir I; Zuev, Yuriy F; Chobert, Jean-Marc; Haertlé, Thomas

    2009-08-01

    As a member of intrinsically unstructured protein family, beta-casein (beta-CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone-like activity in vitro. Like many chaperones, native beta-CN does not contain cysteinyl residues and exhibits strong tendencies for self-association. The chaperone-like activities of three recombinant beta-CNs wild type (WT) beta-CN, C4 beta-CN (with cysteinyl residue in position 4) and C208 beta-CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native beta-CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (beta-CND) of C4-beta-CN and C208 beta-CN were also studied and their chaperone-like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT beta-CN, C208 beta-CN, C4 beta-CN and C4 beta-CND exhibited significantly lower chaperone-like activities than native beta-CN. Dimerization of C208 beta-CN with two distal hydrophilic domains considerably improved its chaperone-like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N-terminal hydrophilic domain as important functional elements in enhancing the chaperone-like activity of native beta-CN. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 623-632, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com. PMID:19322774

  2. Replacement of the C-terminal tetrapeptide (314PAPV317 to 314SSSM317) in interferon regulatory factor-2 alters its N-terminal DNA-binding activity

    Indian Academy of Sciences (India)

    Krishna Prakash; Pramod C Rath

    2010-12-01

    Interferon regulatory factor-2 (IRF-2) is an important transcription factor involved in cell growth regulation, immune response and cancer. IRF-2 can function as a transcriptional repressor and activator depending on its DNA-binding activity and protein–protein interactions. We compared the amino acid sequences of IRF-2 and found a C-terminal tetrapeptide (314PAPV317) of mouse IRF-2 to be different (314SSSM317) from human IRF-2. Recombinant GST-IRF-2 with 314PAPV317 (wild type) and 314SSSM317 (mutant) expressed in Escherichia coli were assessed for DNA-binding activity with 32P-(GAAAGT)4 by electrophoretic mobility shift assay (EMSA). Wild type- and mutant GST-IRF-2 showed similar expression patterns and immunoreactivities but different DNA-binding activities. Mutant (mt) IRF-2 formed higher-molecular-mass, more and stronger DNA–protein complexes in comparison to wild type (wt) IRF-2. Anti-IRF-2 antibody stabilized the DNA–protein complexes formed by both wt IRF-2 and mt IRF-2, resolving the differences. This suggests that PAPV and SSSM sequences at 314-317 in the C-terminal region of mouse and human IRF-2 contribute to conformation of IRF-2 and influence DNA-binding activity of the N-terminal region, indicating intramolecular interactions. Thus, evolution of IRF-2 from murine to human genome has resulted in subtle differences in C-terminal amino acid motifs, which may contribute to qualitative changes in IRF-2-dependent DNA-binding activity and gene expression.

  3. Hydrogen peroxide activates activator protein-1 and mitogen-activated protein kinases in pancreatic stellate cells.

    Science.gov (United States)

    Kikuta, Kazuhiro; Masamune, Atsushi; Satoh, Masahiro; Suzuki, Noriaki; Satoh, Kennichi; Shimosegawa, Tooru

    2006-10-01

    Activated pancreatic stellate cells (PSCs) are implicated in the pathogenesis of pancreatic inflammation and fibrosis, where oxidative stress is thought to play a key role. Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) may act as a second messenger to mediate the actions of growth factors and cytokines. But the role of reactive oxygen species in the activation and regulation of cell functions in PSCs remains largely unknown. We here examined the effects of H(2)O(2) on the activation of signal transduction pathways and cell functions in PSCs. PSCs were isolated from the pancreas of male Wistar rats, and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. Activation of transcription factors was examined by electrophoretic mobility shift assay and luciferase assay. Activation of mitogen-activated protein (MAP) kinases was assessed by Western blotting using anti-phosphospecific antibodies. The effects of H(2)O(2) on proliferation, alpha(1)(I)procollagen gene expression, and monocyte chemoattractant protein-1 production were evaluated. The effect of H(2)O(2) on the transformation of freshly isolated PSCs in culture was also assessed. H(2)O(2) at non-cytotoxic concentrations (up to 100 microM) induced oxidative stress in PSCs. H(2)O(2) activated activator protein-1, but not nuclear factor kappaB. In addition, H(2)O(2) activated three classes of MAP kinases: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAP kinase. H(2)O(2) induced alpha(1)(I)procollagen gene expression but did not induce proliferation or monocyte chemoattractant protein-1 production. H(2)O(2) did not initiate the transformation of freshly isolated PSCs to myofibroblast-like phenotype. Specific activation of these signal transduction pathways and collagen gene expression by H(2)O(2) may play a role in the pathogenesis of pancreatic fibrosis.

  4. The Unstructured N-terminal Region of Arabidopsis Group 4 Late Embryogenesis Abundant (LEA) Proteins Is Required for Folding and for Chaperone-like Activity under Water Deficit.

    Science.gov (United States)

    Cuevas-Velazquez, Cesar L; Saab-Rincón, Gloria; Reyes, José Luis; Covarrubias, Alejandra A

    2016-05-13

    Late embryogenesis abundant (LEA) proteins are a conserved group of proteins widely distributed in the plant kingdom that participate in the tolerance to water deficit of different plant species. In silico analyses indicate that most LEA proteins are structurally disordered. The structural plasticity of these proteins opens the question of whether water deficit modulates their conformation and whether these possible changes are related to their function. In this work, we characterized the secondary structure of Arabidopsis group 4 LEA proteins. We found that they are disordered in aqueous solution, with high intrinsic potential to fold into α-helix. We demonstrate that complete dehydration is not required for these proteins to sample ordered structures because milder water deficit and macromolecular crowding induce high α-helix levels in vitro, suggesting that prevalent conditions under water deficit modulate their conformation. We also show that the N-terminal region, conserved across all group 4 LEA proteins, is necessary and sufficient for conformational transitions and that their protective function is confined to this region, suggesting that folding into α-helix is required for chaperone-like activity under water limitation. We propose that these proteins can exist as different conformers, favoring functional diversity, a moonlighting property arising from their structural dynamics. PMID:27006402

  5. Three new structures of left-handed RADA helical filaments: structural flexibility of N-terminal domain is critical for recombinase activity.

    Science.gov (United States)

    Chang, Yu-Wei; Ko, Tzu-Ping; Lee, Chien-Der; Chang, Yuan-Chih; Lin, Kuei-Ann; Chang, Chia-Seng; Wang, Andrew H-J; Wang, Ting-Fang

    2009-01-01

    RecA family proteins, including bacterial RecA, archaeal RadA, and eukaryotic Dmc1 and Rad51, mediate homologous recombination, a reaction essential for maintaining genome integrity. In the presence of ATP, these proteins bind a single-strand DNA to form a right-handed nucleoprotein filament, which catalyzes pairing and strand exchange with a homologous double-stranded DNA (dsDNA), by as-yet unknown mechanisms. We recently reported a structure of RadA left-handed helical filament, and here present three new structures of RadA left-handed helical filaments. Comparative structural analysis between different RadA/Rad51 helical filaments reveals that the N-terminal domain (NTD) of RadA/Rad51, implicated in dsDNA binding, is highly flexible. We identify a hinge region between NTD and polymerization motif as responsible for rigid body movement of NTD. Mutant analysis further confirms that structural flexibility of NTD is essential for RadA's recombinase activity. These results support our previous hypothesis that ATP-dependent axial rotation of RadA nucleoprotein helical filament promotes homologous recombination.

  6. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

    Science.gov (United States)

    Harris, Samantha P; Belknap, Betty; Van Sciver, Robert E; White, Howard D; Galkin, Vitold E

    2016-02-01

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere.

  7. Degradation of Activated Protein Kinases by Ubiquitination

    OpenAIRE

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases.

  8. Crystal Structure of Full-length Mycobacterium tuberculosis H37Rv Glycogen Branching Enzyme; Insights of N-Terminal [beta]-Sandwich in Sustrate Specifity and Enzymatic Activity

    Energy Technology Data Exchange (ETDEWEB)

    Pal, Kuntal; Kumar, Shiva; Sharma, Shikha; Garg, Saurabh Kumar; Alam, Mohammad Suhail; Xu, H. Eric; Agrawal, Pushpa; Swaminathan, Kunchithapadam (NU Sinapore); (Van Andel); (IMT-India)

    2010-07-13

    The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an {alpha}-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1 {yields} 4 bond and making a new 1 {yields} 6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-{angstrom} resolution. MtbGlgBWT contains four domains: N1 {beta}-sandwich, N2 {beta}-sandwich, a central ({beta}/{alpha}){sub 8} domain that houses the catalytic site, and a C-terminal {beta}-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) Mtb{Delta}108GlgB protein. The N1 {beta}-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 {beta}-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and Mtb{Delta}108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1 {yields} 4 bond breakage) and isomerization (1 {yields} 6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and Mtb{Delta}108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (EC{Delta}112GlgB).

  9. Chimeric RXFP1 and RXFP2 receptors highlight the similar mechanism of activation utilizing their N-terminal low density lipoprotein class A modules

    Directory of Open Access Journals (Sweden)

    Shoni eBruell

    2013-11-01

    Full Text Available Relaxin family peptide (RXFP receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low density lipoprotein type A (LDLa module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP based signalling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a

  10. Chimeric RXFP1 and RXFP2 Receptors Highlight the Similar Mechanism of Activation Utilizing Their N-Terminal Low-Density Lipoprotein Class A Modules.

    Science.gov (United States)

    Bruell, Shoni; Kong, Roy C K; Petrie, Emma J; Hoare, Brad; Wade, John D; Scott, Daniel J; Gooley, Paul R; Bathgate, Ross A D

    2013-01-01

    Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low-density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP-based signaling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand-mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a similar mechanism. PMID

  11. Functional characterization of human RSK4, a new 90-kDa ribosomal S6 kinase, reveals constitutive activation in most cell types

    DEFF Research Database (Denmark)

    Dümmler, Bettina A; Hauge, Camilla; Silber, Joachim;

    2005-01-01

    The 90-kDa ribosomal S6 kinases (RSK1-3) are important mediators of growth factor stimulation of cellular proliferation, survival, and differentiation and are activated via coordinated phosphorylation by ERK and 3-phosphoinositide-dependent protein kinase-1 (PDK1). Here we performed the functional...... for phosphorylation of Ser232, a key regulatory site in the activation loop of the N-terminal kinase domain, that in other RSKs is phosphorylated by PDK1. The unusual regulation and growth factor-independent kinase activity indicate that RSK4 is functionally distinct from other RSKs and may help explain recent...

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

    DEFF Research Database (Denmark)

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

    2000-01-01

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

  13. Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

    Institute of Scientific and Technical Information of China (English)

    Sergiy; Kostenko; Gianina; Dumitriu; Kari; Jenssen; Lgreid; Ugo; Moens

    2011-01-01

    Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

  14. Construction and identification of dominant-negative c-Jun N-terminal kinase(DN-JNK)recombinant adenovirus%DN-JNK基因重组腺病毒的构建和鉴定

    Institute of Scientific and Technical Information of China (English)

    张佳妮; 刘慧霞; 陈金虎; 郭敏; 全养雅; 谭莺

    2009-01-01

    Objective To construct and identify replication deficient recombinant adenovirus expressing human c-Jun N-terminal kinase(JNK)by homologous recombination adenovirus dominant-negative type JNK(Ad-DN-JNK).Methods The linearized recombinant shuttle vector pAdTrack-CMV-DN-JNK Was co-transformed with backbone vector pAdEasy-l into bacteria BJ5183 for recombinant adenoviral vector.The recombinant adenoviral vector was transfected into HEK293 packing cells tO construct replication deficient recombinant adenovirus,and then the recombinant edenovirns WaS detected by PCR and DNA sequencing.Western blot analysis was utilized to detect the Cxpression of Ad-DN-JNK and the level of insulin receptor substrate l Serine307 phosphorylation.Results JNK recombinant adenoviral vectorcould be effectively transfeeted into HEK 293 cell and successfully packed by intracellular enzyme.The expression of green fluorescent protein(GFP)Was observed on the 5th day after transfection.The fragment of JNK gene waS amplified by PCR and identified by sequencing.The titer of the prepared Ad-DN-JNK is 2.5×1010 pfu/ml.The animal experiment confirmed that constructed Ad-DN-JNK could be effectively expressed in liver tissue.Conclusion The research successfully constructed recombinant adenoviral vector and recombinant adenoviral particle.Animal experiment demonstrated the Ad-DN-JNK could effectively mediated the expression of DN-JNK gene and down-regulated the level of IRSlscfine307 phosphorylation.The achievement laid a foundation for further investigation of the function and application of JNK.%目的 制备表达人c-jun氨基末端激酶(JNK)复制缺陷型重组腺病毒(Ad-DN-JNK).并通过动物实验进行功能鉴定.方法 将重组穿梭载体pAdTraek-CMV-DN-JNK线性化后,与pAdEasy-1共转化大肠杆菌BJ5138,进行同源重组得到重组腺病毒载体.将重组腺病毒栽体转染入包装细胞HEK293内制备复制缺陷型重组腺病毒,并经PCR及DNA测序鉴定.Western blot检

  15. Antiepileptic Effect of Uncaria rhynchophylla and Rhynchophylline Involved in the Initiation of c-Jun N-Terminal Kinase Phosphorylation of MAPK Signal Pathways in Acute Seizures of Kainic Acid-Treated Rats

    Directory of Open Access Journals (Sweden)

    Hsin-Cheng Hsu

    2013-01-01

    Full Text Available Seizures cause inflammation of the central nervous system. The extent of the inflammation is related to the severity and recurrence of the seizures. Cell surface receptors are stimulated by stimulators such as kainic acid (KA, which causes intracellular mitogen-activated protein kinase (MAPK signal pathway transmission to coordinate a response. It is known that Uncaria rhynchophylla (UR and rhynchophylline (RP have anticonvulsive effects, although the mechanisms remain unclear. Therefore, the purpose of this study is to develop a novel strategy for treating epilepsy by investigating how UR and RP initiate their anticonvulsive mechanisms. Sprague-Dawley rats were administered KA (12 mg/kg, i.p. to induce seizure before being sacrificed. The brain was removed 3 h after KA administration. The results indicate that pretreatment with UR (1.0 g/kg, RP (0.25 mg/kg, and valproic acid (VA, 250 mg/kg for 3 d could reduce epileptic seizures and could also reduce the expression of c-Jun aminoterminal kinase phosphorylation (JNKp of MAPK signal pathways in the cerebral cortex and hippocampus brain tissues. Proinflammatory cytokines interleukin (IL-1β, IL-6, and tumor necrosis factor-α remain unchanged, indicating that the anticonvulsive effect of UR and RP is initially involved in the JNKp MAPK signal pathway during the KA-induced acute seizure period.

  16. N-Terminal Region of GbIspH1, Ginkgo biloba IspH Type 1, May Be Involved in the pH-Dependent Regulation of Enzyme Activity

    Directory of Open Access Journals (Sweden)

    Bok-Kyu Shin

    2015-01-01

    Full Text Available GbIspH1, IspH type 1 in Ginkgo biloba chloroplast, is the Fe/S enzyme catalyzing the reductive dehydroxylation of HMBPP to isopentenyl diphosphate (IPP and dimethylallyl diphosphate (DMAPP at the final step of methylerythritol phosphate pathway in chloroplast. Compared to the bacterial IspH, plant IspH, including GbIspH1, has an additional polypeptide chain at the N-terminus. Here, biochemical function of the N-terminal region of GbIspH1 was investigated with the N-terminal truncated GbIspH1 (GbIspH1-truncated. Both wild type GbIspH1 (GbIspH1-full and GbIspH1-truncated were catalytically active and produced IPP and DMAPP in a ratio of 15 : 1. Kinetic parameters of KM (17.3 ± 1.9 and 14.9 ± 2.3 µM and kcat (369 ± 10 and 347 ± 12 min−1 at pH 8.0 were obtained for GbIspH1-full and GbIspH1-truncated, respectively. Interestingly, GbIspH1-full and GbIspH1-truncated showed significantly different pH-dependent activities, and the maximum enzyme activities were obtained at pH 8.0 and 7.5, respectively. However, catalytic activation energies (Ea of GbIspH1-full and GbIspH1-truncated were almost the same with 36.5 ± 1.6 and 35.0 ± 1.9 kJ/mol, respectively. It was suggested that the N-terminal region of GbIspH1 is involved in the pH-dependent regulation of enzyme activity during photosynthesis.

  17. MEK kinase 1 activity is required for definitive erythropoiesis in the mouse fetal liver

    DEFF Research Database (Denmark)

    Bonnesen, Barbara; Ørskov, Cathrine; Rasmussen, Susanne;

    2005-01-01

    KD) embryos have normal morphology but are anemic due to failure of definitive erythropoiesis. When Mekk1(DeltaKD) fetal liver cells were transferred to lethally irradiated wild-type hosts, mature red blood cells were generated from the mutant cells, suggesting that MEKK1 functions in a non-cell......Mitogen-activated protein kinase/extracellular signal to regulated kinase (MEK) kinase 1 (MEKK1) is a c-Jun N-terminal kinase (JNK) activating kinase known to be implicated in proinflammatory responses and cell motility. Using mice deficient for MEKK1 kinase activity (Mekk1(DeltaKD)) we show a role...... for MEKK1 in definitive mouse erythropoiesis. Although Mekk1(DeltaKD) mice are alive and fertile on a 129 x C57/BL6 background, the frequency of Mekk1(DeltaKD) embryos that develop past embryonic day (E) 14.5 is dramatically reduced when backcrossed into the C57/BL6 background. At E13.5, Mekk1(Delta...

  18. Analysis of proteolytic processes and enzymatic activities in the generation of huntingtin n-terminal fragments in an HEK293 cell model.

    Directory of Open Access Journals (Sweden)

    Andrew T N Tebbenkamp

    Full Text Available BACKGROUND: N-terminal fragments of mutant huntingtin (htt that terminate between residues 90-115, termed cleavage product A or 1 (cp-A/1, form intracellular and intranuclear inclusion bodies in the brains of patients with Huntington's disease (HD. These fragments appear to be proteolytic products of the full-length protein. Here, we use an HEK293 cell culture model to investigate huntingtin proteolytic processing; previous studies of these cells have demonstrated cleavage of htt to cp-A/1 like htt fragments. RESULTS: Recombinant N-terminal htt fragments, terminating at residue 171 (also referred to as cp-B/2 like, were efficiently cleaved to produce cp-A/1 whereas fragments representing endogenous caspase, calpain, and metalloproteinase cleavage products, terminating between residues 400-600, were inefficiently cleaved. Using cysteine-labeling techniques and antibody binding mapping, we localized the C-terminus of the cp-A/1 fragments produced by HEK293 cells to sequences minimally limited by cysteine 105 and an antibody epitope composed of residues 115-124. A combination of genetic and pharmacologic approaches to inhibit potential proteases, including γ-secretase and calpain, proved ineffective in preventing production of cp-A/1. CONCLUSIONS: Our findings indicate that HEK293 cells express a protease that is capable of efficiently cleaving cp-B/2 like fragments of htt with normal or expanded glutamine repeats. For reasons that remain unclear, this protease cleaves longer htt fragments, with normal or expanded glutamine expansions, much less efficiently. The protease in HEK293 cells that is capable of generating a cp-A/1 like htt fragment may be a novel protease with a high preference for a cp-B/2-like htt fragment as substrate.

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

    Directory of Open Access Journals (Sweden)

    Yun-Hee Choi

    2015-11-01

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

  20. Glutathione S-transferases interact with AMP-activated protein kinase: evidence for S-glutathionylation and activation in vitro.

    Science.gov (United States)

    Klaus, Anna; Zorman, Sarah; Berthier, Alexandre; 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.

  1. The membranotropic activity of N-terminal peptides from the pore-forming proteins sticholysin I and II is modulated by hydrophobic and electrostatic interactions as well as lipid composition

    Indian Academy of Sciences (India)

    Uris Ros; Lohans Pedrera; Daylín Díaz; Juan C De Karam; Tatiane P Sudbrack; Pedro A Valiente; Diana Martínez; Eduardo M Cilli; Fabiola Pazos; Rosangela Itri; Maria E Lanio; Shirley Schreier; Carlos Álvarez

    2011-12-01

    The sea anemone Stichodactyla helianthus produces two pore-forming proteins, sticholysins I and II (St I and St II). Despite their high identity (93%), these toxins exhibit differences in hemolytic activity that can be related to those found in their N-terminal. To clarify the contribution of the N-terminal amino acid residues to the activity of the toxins, we synthesized peptides spanning residues 1–31 of St I (StI1-31) or 1–30 of St II (StII1-30) and demonstrated that StII1-30 promotes erythrocyte lysis to a higher extent than StI1-31. For a better understanding of the molecular mechanism underlying the peptide activity, here we studied their binding to lipid monolayers and pemeabilizing activity in liposomes. For this, we examined the effect on peptide membranotropic activity of including phospatidic acid and cholesterol in a lipid mixture of phosphatidylcholine and sphingomyelin. The results suggest the importance of continuity of the 1–10 hydrophobic sequence in StII1-30 for displaying higher binding and activity, in spite of both peptides’ abilities to form pores in giant unilamellar vesicles. Thus, the different peptide membranotropic action is explained in terms of the differences in hydrophobic and electrostatic peptide properties as well as the enhancing role of membrane inhomogeneities.

  2. EhPAK2, a novel p21-activated kinase, is required for collagen invasion and capping in Entamoeba histolytica.

    Science.gov (United States)

    Arias-Romero, Luis Enrique; de Jesús Almáraz-Barrera, Ma; Díaz-Valencia, Juan Daniel; Rojo-Domínguez, Arturo; Hernandez-Rivas, Rosaura; Vargas, Miguel

    2006-09-01

    p21-activated kinases (PAKs) are a highly conserved family of enzymes that are activated by Rho GTPases. All PAKs contain an N-terminal Cdc42/Rac interacting binding (CRIB) domain, which confers binding to these GTPases, and a C-terminal kinase domain. In addition, some PAKs such as Cla4p, Skm1p and Pak2p contain an N-terminal pleckstrin homology (PH) domain and form a distinct group of PAK proteins involved in cell morphology, cell-cycle and gene transcription. Here, we describe a novel p21-activated kinase, denominated EhPAK2, on the parasitic protozoan Entamoeba histolytica. This is the first reported Entamoeba PAK member that contains a N-terminal PH domain and a highly conserved CRIB domain. EhPAK2 CRIB domain shares 29% of amino acid identity and 53% of amino acid homology with these of DdPAKC from Dictyostelium discoideum and Cla4p from Saccharomyces cerevisiae and binds in vitro and in vivo to EhRacA GTPase. This domain also possesses the conserved residues His123, Phe134 and Trp141, which are important for the interaction with the effector loop and strand beta2 of the GTPase; and the residues Met121 and Phe145, which are specific for the interaction of EhPAK2 with EhRacA. Functional studies of EhPAK2 showed that its C-terminal kinase domain had activity toward myelin basic protein. Cellular studies showed that Entamoeba trophozoites transfected with the vector pExEhNeo/kinase-myc, had a 90% decrease in the ability to invade a collagen matrix as well as severe defects in capping, suggesting the involvement of EhPAK2 in these cellular processes.

  3. The Na+/H+ exchanger, NHE1, differentially regulates mitogen-activated protein kinase subfamilies after osmotic shrinkage in Ehrlich Lettre Ascites cells

    DEFF Research Database (Denmark)

    Petersen, Stine Helene Falsig; Rasmussen, Maria; Darborg, Barbara Vasek;

    2007-01-01

    Osmotic stress modulates mitogen activated protein kinase (MAPK) activities, leading to altered gene transcription and cell death/survival balance, however, the mechanisms involved are incompletely elucidated. Here, we show, using a combination of biochemical and molecular biology approaches......, that three MAPKs exhibit unique interrelationships with the Na(+)/H(+) exchanger, NHE1, after osmotic cell shrinkage: Extracellular Signal Regulated Kinase (ERK1/2) is inhibited in an NHE1-dependent, pH(i)-independent manner, c-Jun N-terminal kinase (JNK1/2) is stimulated, in part through NHE1-mediated...

  4. Antimicrobial activity of human α-defensin 5 and its linear analogs: N-terminal fatty acylation results in enhanced antimicrobial activity of the linear analogs.

    Science.gov (United States)

    Mathew, Basil; Nagaraj, Ramakrishnan

    2015-09-01

    Human α-defensin 5 (HD5) exhibits broad spectrum antimicrobial activity and plays an important role in mucosal immunity of the small intestine. Although there have been several studies, the structural requirements for activity and mechanism of bacterial killing is yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD5 and linear analogs. Cysteine deletions attenuated the antibacterial activity considerably. Candidacidal activity was affected to a lesser extent. Fatty acid conjugated linear analogs showed antimicrobial activity comparable activity to HD5. Effective surface charge neutralization of bacteria was observed for HD5 as compared to the non-fatty acylated linear analogs. Our results show that HD5 and non-fatty acylated linear analogs enter the bacterial cytoplasm without causing damage to the bacterial inner membrane. Although fatty acylated peptides exhibited antimicrobial activity comparable to HD5, their mechanism of action involved permeabilization of the Escherichia coli inner membrane. HD5 and analogs had the ability to bind plasmid DNA. HD5 had greater binding affinity to plasmid DNA as compared to the analogs. The three dimensional structure of HD5 favors greater interaction with the bacterial cell surface and also with DNA. Antibacterial activity of HD5 involves entry into bacterial cytoplasm and binding to DNA which would result in shut down of the bacterial metabolism leading to cell death. We show how a moderately active linear peptide derived from the α-defensin HD5 can be engineered to enhance antimicrobial activity almost comparable to the native peptide. PMID:26206286

  5. Multifunctional roles for the N-terminal basic motif of Alfalfa mosaic virus coat protein: nucleolar/cytoplasmic shuttling, modulation of RNA-binding activity, and virion formation.

    Science.gov (United States)

    Herranz, Mari Carmen; Pallas, Vicente; Aparicio, Frederic

    2012-08-01

    In addition to virion formation, the coat protein (CP) of Alfalfa mosaic virus (AMV) is involved in the regulation of replication and translation of viral RNAs, and in cell-to-cell and systemic movement of the virus. An intriguing feature of the AMV CP is its nuclear and nucleolar accumulation. Here, we identify an N-terminal lysine-rich nucleolar localization signal (NoLS) in the AMV CP required to both enter the nucleus and accumulate in the nucleolus of infected cells, and a C-terminal leucine-rich domain which might function as a nuclear export signal. Moreover, we demonstrate that AMV CP interacts with importin-α, a component of the classical nuclear import pathway. A mutant AMV RNA 3 unable to target the nucleolus exhibited reduced plus-strand RNA synthesis and cell-to-cell spread. Moreover, virion formation and systemic movement were completely abolished in plants infected with this mutant. In vitro analysis demonstrated that specific lysine residues within the NoLS are also involved in modulating CP-RNA binding and CP dimerization, suggesting that the NoLS represents a multifunctional domain within the AMV CP. The observation that nuclear and nucleolar import signals mask RNA-binding properties of AMV CP, essential for viral replication and translation, supports a model in which viral expression is carefully modulated by a cytoplasmic/nuclear balance of CP accumulation. PMID:22746826

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

    Science.gov (United States)

    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

  7. Thymic Stromal Lymphopoietin Promotes Fibrosis and Activates Mitogen-Activated Protein Kinases in MRC-5 Cells.

    Science.gov (United States)

    Li, Li; Tang, Su; Tang, Xiaodong

    2016-01-01

    BACKGROUND Acute lung injury (ALI) is a life-threatening hypoxemic respiratory disorder with high incidence and mortality. ALI usually manifests as widespread inflammation and lung fibrosis with the accumulation of pro-inflammatory and pro-fibrotic factors and collagen. Thymic stromal lymphopoietin (TSLP) has a significant role in regulation of inflammation but little is known about its roles in lung fibrosis or ALI. This study aimed to define the role and possible regulatory mechanism of TSLP in lung fibrosis. MATERIAL AND METHODS We cultured human lung fibroblast MRC-5 cells and overexpressed or inhibited TSLP by the vector or small interfering RNA transfection. Then, the pro-fibrotic factors skeletal muscle actin alpha (α-SMA) and collagen I, and the 4 mitogen-activated protein kinases (MAPKs) - MAPK7, p38, extracellular signal-regulated kinase 1 (ERK1), and c-Jun N-terminal kinase 1 (JNK1) - were detected by Western blot. RESULTS Results showed that TSLP promoted the production of α-SMA and collagen I (Pmechanism of fibrosis. PMID:27385084

  8. MED14 tethers mediator to the N-terminal domain of peroxisome proliferator-activated receptor gamma and is required for full transcriptional activity and adipogenesis

    DEFF Research Database (Denmark)

    Grøntved, Lars; Madsen, Maria S; Boergesen, Michael;

    2010-01-01

    of PPARgamma in a ligand-independent manner. Interestingly, MED14 knockdown does not affect the recruitment of PPARgamma, MED6, and MED8 to the Fabp4 enhancer but does reduce their occupancy of the Fabp4 proximal promoter. In agreement with the necessity of MED14 for PPARgamma transcriptional activity, we show...

  9. Components of the mitogen-activated protein kinase cascade are activated in hepatic cells by Echinococcus multilocularis metacestode

    Institute of Scientific and Technical Information of China (English)

    Ren-Yong Lin; Jun-Hua Wang; Xiao-Mei Lu; Xiao-Tao Zhou; Georges Mantion; Hao Wen; Dominique A Vuitton; Lysiane Richert

    2009-01-01

    AIM: To explore the ef fec t of Echinococ cus multilocularis on the activation of mitogen-activated protein kinase (MAPK) signaling pathways and on liver cell proliferation. METHODS: Changes in the phosphorylation of MAPKs and proliferating cell nuclear antigen (PCNA) expression were measured in the liver of patients with alveolar echinococcosis (AE). MAPKs, MEK1/2 [MAPK/ extracellular signal-regulated protein kinase (ERK) kinase] and ribosomal S6 kinase (RSK) phosphorylation were detected in primary cultures of rat hepatocytes in contact in vitro with (1) E. multilocularis vesicle fluid (EmF), (2) E. multilocularis-conditioned medium (EmCM). RESULTS: In the liver of AE patients, ERK 1/2 and p38 MAPK were activated and PCNA expression was increased, especially in the vicinity of the metacestode. Upon exposure to EmF, p38, c-Jun N-terminal kinase (JNK) and ERK1/2 were also activated in hepatocytes in vitro, as well as MEK1/2 and RSK, in the absence of any toxic effect. Upon exposure to EmCM, only JNK was up-regulated. CONCLUSION: Previous studies have demonstrated an influence of the host on the MAPK cascade in E. multilocularis. Our data suggest that the reverse, i.e. parasite-derived signals efficiently acting on MAPK signaling pathways in host liver cells, is actually operating.

  10. Role of the N-terminal activation domain of coactivator CoCoA in mediating transcriptional activation by β-catenin*

    OpenAIRE

    Yang, Catherine K.; Kim, Jeong Hoon; Stallcup, Michael R.

    2006-01-01

    The coiled-coil coactivator (CoCoA) is involved in transcriptional activation of target genes by nuclear receptors and the xenobiotic aryl hydrocarbon receptor, as well as target genes of the Wnt signaling pathway, which is mediated by the lymphocyte enhancer factor (LEF)/T cell factor transcription factors and the coactivator β-catenin. The recruitment of CoCoA by nuclear receptors is accomplished by the interaction of the central coiled-coiled domain of CoCoA with p160 coactivators; the C-t...

  11. Serine phosphorylation of NPM-ALK, which is dependent on the auto-activation of the kinase activation loop, contributes to its oncogenic potential.

    Science.gov (United States)

    Wang, Peng; Wu, Fang; Zhang, Jingdong; McMullen, Todd; Young, Leah C; Ingham, Robert J; Li, Liang; Lai, Raymond

    2011-02-01

    It is well established that the tumorigenic potential of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), an oncogenic tyrosine kinase, is dependent on its tyrosine phosphorylation. Using tandem affinity purification-mass spectrometry, we found evidence of phosphorylation of three serine residues of NPM-ALK (Serine¹³⁵, Serine¹⁶⁴ and Serine⁴⁹⁷) ectopically expressed in GP293 cells. Using a specific anti-phosphoserine antibody and immunoprecipitation, we confirmed the presence of serine phosphorylation of NPM-ALK in all three NPM-ALK-expressing cell lines examined. Similar to the tyrosine phosphorylation, phosphorylation of these serine residues was dependent on the activation status of the kinase activation loop of ALK. All of these three serine residues are biologically important as mutation of any one of these residues resulted in a significant reduction in the tumorigenicity of NPM-ALK (assessed by cell viability and clonogenic assay), which correlated with a substantial reduction in the phosphorylation of extracellular signal-regulated kinase 1/2, c-jun N-terminal kinase and signal transducer and activator of transcription 6. Serine phosphorylation of NPM-ALK appears to be regulated by multiple serine kinases since it was markedly reduced by pharmacologic inhibitors for glycogen synthase kinase-3, casein kinase I or mitogen-activated protein kinases. In summary, our study is the first to identify serine phosphorylation of NPM-ALK and to provide evidence that it enhances the tumorigenic potential of this oncogenic protein.

  12. Cyclin H binding to the RARα activation function (AF)-2 domain directs phosphorylation of the AF-1 domain by cyclin-dependent kinase 7

    OpenAIRE

    Bour, Gaétan; Gaillard, Emilie; Bruck, Nathalie; Lalevée, Sébastien; Plassat, Jean-Luc; Busso, Didier; Samama, Jean-Pierre; Rochette-Egly, Cécile

    2005-01-01

    The transcriptional activity of nuclear retinoic acid receptors (RARs), which act as RAR/retinoid X receptor (RXR) heterodimers, depends on two activation functions, AF-1 and AF-2, which are targets for phosphorylations and synergize for the activation of retinoic acid target genes. The N-terminal AF-1 domain of RARα is phosphorylated at S77 by the cyclin-dependent kinase (cdk)-activating kinase (CAK) subcomplex (cdk7/cyclin H/MAT1) of the general transcription factor TFIIH. Here, we show tha...

  13. 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

    Science.gov (United States)

    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.

  14. Crosstalk and signalling switches in mitogen-activated protein kinase cascades

    Directory of Open Access Journals (Sweden)

    Dirk eFey

    2012-09-01

    Full Text Available Mitogen-activated protein kinase (MAPK cascades control cell fate decisions, such as proliferation, differentiation and apoptosis by integrating and processing intra- and extracellular cues. However, similar MAPK kinetic profiles can be associated with opposing cellular decisions depending on cell type, signal strength and dynamics. This implies that signalling by each individual MAPK cascade has to be considered in the context of the entire MAPK network. Here, we develop a dynamic model of feedback and crosstalk for the three major MAPK cascades; extracellular signal-regulated kinase (ERK, p38 mitogen-activated protein kinase (p38, c-Jun N-terminal kinase (JNK, and also include input from protein kinase B (AKT. Focusing on the bistable activation characteristics of the JNK pathway, this model explains how pathway crosstalk harmonises different MAPK responses resulting in pivotal cell fate decisions. We show that JNK can switch from a transient to sustained activity due to multiple positive feedback loops. Once activated, positive feedback locks JNK in a highly active state and promotes cell death. The switch is modulated by the ERK, p38 and AKT pathways. ERK activation enhances the dual specificity phosphatase (DUSP mediated dephosphorylation of JNK and shifts the threshold of the apoptotic switch to higher inputs. Activation of p38 restores the threshold by inhibiting ERK activity via the PP1 or PP2A phosphatases. Finally, AKT activation inhibits the JNK positive feedback, thus abrogating the apoptotic switch and allowing only proliferative signalling. Our model facilitates understanding of how cancerous deregulations disturb MAPK signal processing and provides explanations for certain drug resistances. We highlight a critical role of DUSP1 and DUSP2 expression patterns in facilitating the switching of JNK activity and show how oncogene induced ERK hyperactivity prevents the normal apoptotic switch explaining the failure ocertain drugs to

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-03-02

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

  16. Site-specific phosphorylation of the DNA damage response mediator rad9 by cyclin-dependent kinases regulates activation of checkpoint kinase 1.

    Directory of Open Access Journals (Sweden)

    Carla Manuela Abreu

    2013-04-01

    Full Text Available The mediators of the DNA damage response (DDR are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR-specific protein kinases.

  17. The metalloid arsenite induces nuclear export of Id3 possibly via binding to the N-terminal cysteine residues

    Energy Technology Data Exchange (ETDEWEB)

    Kurooka, Hisanori, E-mail: hkurooka@u-fukui.ac.jp [Division of Molecular Genetics, Department of Biochemistry and Bioinformative Sciences, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Research and Education Program for Life Science, University of Fukui, Fukui (Japan); Sugai, Manabu [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto (Japan); Mori, Kentaro [Division of Molecular Genetics, Department of Biochemistry and Bioinformative Sciences, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Yokota, Yoshifumi, E-mail: yokota@u-fukui.ac.jp [Division of Molecular Genetics, Department of Biochemistry and Bioinformative Sciences, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Research and Education Program for Life Science, University of Fukui, Fukui (Japan)

    2013-04-19

    Highlights: •Sodium arsenite induces cytoplasmic accumulation of Id3. •Arsenite binds to closely spaced N-terminal cysteine residues of Id3. •N-terminal cysteines are essential for arsenite-induced nuclear export of Id3. •Nuclear export of Id3 counteracts its transcriptional repression activity. -- Abstract: Ids are versatile transcriptional repressors that regulate cell proliferation and differentiation, and appropriate subcellular localization of the Id proteins is important for their functions. We previously identified distinct functional nuclear export signals (NESs) in Id1 and Id2, but no active NES has been reported in Id3. In this study, we found that treatment with the stress-inducing metalloid arsenite led to the accumulation of GFP-tagged Id3 in the cytoplasm. Cytoplasmic accumulation was impaired by a mutation in the Id3 NES-like sequence resembling the Id1 NES, located at the end of the HLH domain. It was also blocked by co-treatment with the CRM1-specific nuclear export inhibitor leptomycin B (LMB), but not with the inhibitors for mitogen-activated protein kinases (MAPKs). Importantly, we showed that the closely spaced N-terminal cysteine residues of Id3 interacted with the arsenic derivative phenylarsine oxide (PAO) and were essential for the arsenite-induced cytoplasmic accumulation, suggesting that arsenite induces the CRM1-dependent nuclear export of Id3 via binding to the N-terminal cysteines. Finally, we demonstrated that Id3 significantly repressed arsenite-stimulated transcription of the immediate-early gene Egr-1 and that this repression activity was inversely correlated with the arsenite-induced nuclear export. Our results imply that Id3 may be involved in the biological action of arsenite.

  18. The metalloid arsenite induces nuclear export of Id3 possibly via binding to the N-terminal cysteine residues

    International Nuclear Information System (INIS)

    Highlights: •Sodium arsenite induces cytoplasmic accumulation of Id3. •Arsenite binds to closely spaced N-terminal cysteine residues of Id3. •N-terminal cysteines are essential for arsenite-induced nuclear export of Id3. •Nuclear export of Id3 counteracts its transcriptional repression activity. -- Abstract: Ids are versatile transcriptional repressors that regulate cell proliferation and differentiation, and appropriate subcellular localization of the Id proteins is important for their functions. We previously identified distinct functional nuclear export signals (NESs) in Id1 and Id2, but no active NES has been reported in Id3. In this study, we found that treatment with the stress-inducing metalloid arsenite led to the accumulation of GFP-tagged Id3 in the cytoplasm. Cytoplasmic accumulation was impaired by a mutation in the Id3 NES-like sequence resembling the Id1 NES, located at the end of the HLH domain. It was also blocked by co-treatment with the CRM1-specific nuclear export inhibitor leptomycin B (LMB), but not with the inhibitors for mitogen-activated protein kinases (MAPKs). Importantly, we showed that the closely spaced N-terminal cysteine residues of Id3 interacted with the arsenic derivative phenylarsine oxide (PAO) and were essential for the arsenite-induced cytoplasmic accumulation, suggesting that arsenite induces the CRM1-dependent nuclear export of Id3 via binding to the N-terminal cysteines. Finally, we demonstrated that Id3 significantly repressed arsenite-stimulated transcription of the immediate-early gene Egr-1 and that this repression activity was inversely correlated with the arsenite-induced nuclear export. Our results imply that Id3 may be involved in the biological action of arsenite

  19. A novel viral thymidylate kinase with dual kinase activity.

    Science.gov (United States)

    Guevara-Hernandez, Eduardo; Arvizu-Flores, Aldo A; Lugo-Sanchez, Maria E; Velazquez-Contreras, Enrique F; Castillo-Yañez, Francisco J; Brieba, Luis G; Sotelo-Mundo, Rogerio R

    2015-10-01

    Nucleotide phosphorylation is a key step in DNA replication and viral infections, since suitable levels of nucleotide triphosphates pool are required for this process. Deoxythymidine monophosphate (dTMP) is produced either by de novo or salvage pathways, which is further phosphorylated to deoxythymidine triphosphate (dTTP). Thymidyne monophosphate kinase (TMK) is the enzyme in the junction of both pathways, which phosphorylates dTMP to yield deoxythymidine diphosphate (dTDP) using adenosine triphosphate (ATP) as a phosphate donor. White spot syndrome virus (WSSV) genome contains an open reading frame (ORF454) that encodes a thymidine kinase and TMK domains in a single polypeptide. We overexpressed the TMK ORF454 domain (TMKwssv) and its specific activity was measured with dTMP and dTDP as phosphate acceptors. We found that TMKwssv can phosphorylate dTMP to yield dTDP and also is able to use dTDP as a substrate to produce dTTP. Kinetic parameters K M and k cat were calculated for dTMP (110 μM, 3.6 s(-1)), dTDP (251 μM, 0.9 s(-1)) and ATP (92 μM, 3.2 s(-1)) substrates, and TMKwssv showed a sequential ordered bi-bi reaction mechanism. The binding constants K d for dTMP (1.9 μM) and dTDP (10 μM) to TMKwssv were determined by Isothermal Titration Calorimetry. The affinity of the nucleotidic analog stavudine monophosphate was in the same order of magnitude (K d 3.6 μM) to the canonical substrate dTMP. These results suggest that nucleotide analogues such as stavudine could be a suitable antiviral strategy for the WSSV-associated disease.

  20. Molecular Imaging of the ATM Kinase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Terence M. [Department of Radiation Oncology, Ohio State University, Columbus, Ohio (United States); Nyati, Shyam [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Ross, Brian D. [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Rehemtulla, Alnawaz, E-mail: alnawaz@umich.edu [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)

    2013-08-01

    Purpose: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. Methods and Materials: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. Results: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlated in a time-dependent fashion with changes in Chk2 activity. Conclusions: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.

  1. Activation of extracellular signal-regulated kinase during silibinin-protected, isoproterenol-induced apoptosis in rat cardiac myocytes is tyrosine kinase pathway-mediated and protein kinase C-dependent

    Institute of Scientific and Technical Information of China (English)

    Bei ZHOU; Li-jun WU; Shin-ichi TASHIRO; Satoshi ONODERA; Fumiaki UCHIUMI; Takashi IKEJIMA

    2007-01-01

    Aim: To investigate the mechanism of silibinin-protected isoproterenol-induced apoptosis in rat cardiac myocytes.Methods: The viability of rat cardiac myocytes was measured by MTT method. The apoptotic ratio was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Protein kinase C (PKC) activity assay was carried out according to the instructions of the PepTag non-radioactive protein kinase C assay kit. Western blot analysis was used to evaluate the level of Ras, Raf-1 and mitogen-activated protein kinase (MAPK) expression.Results: The protective effects of silibinin were significantly sup-pressed by inhibitors, including genistein, manumycin A and GW5074 [inhibitors for protein tyrosine kinases (PTK), Ras and Raf- 1, respectively]. The exposure of rat cardiac myocytes to isoproterenol alone caused decreased PKC activity, which was prevented by pretreatment with silibinin dose-dependently. Simultaneously,the increased expression of Ras and Raf-1 activated by silibinin were blocked by the PKC inhibitor, stauroporine. In addition, the extracellularly responsive kinase (ERK) inhibitor, PD98059, suppressed silibinin-protected apoptosis, whereas the p38 MAPK inhibitor, SB203580, protected cardiac myocytes from isoproterenol-induced injury, and the c-Jun N-terminal kinase (JNK) inhibitor, SP600125 had no protective effects. Furthermore, Western blot analysis showed that the expres-sion of phosphorylated ERK was increased by silibinin, the expression of phos-phorylated p38 MAPK was decreased and total ERK, p38, JNK and phosphory-lated JNK MAPK did not change after treatment with both isoproterenol and silibinin. Furthermore, pretreatment of cardiac myocyte with PKC, Ras and Raf inhibitors significantly blocked ERK phosphorylation.Conclusion: Silibinin is suggested to protect isoproterenol-induced rat cardiac myocyte apoptosis by activating the tyrosine kinase pathway, PKC and MAPK pathways.

  2. Adipocyte lipolysis-stimulated interleukin-6 production requires sphingosine kinase 1 activity.

    Science.gov (United States)

    Zhang, Wenliang; Mottillo, Emilio P; Zhao, Jiawei; Gartung, Allison; VanHecke, Garrett C; Lee, Jen-Fu; Maddipati, Krishna R; Xu, Haiyan; Ahn, Young-Hoon; Proia, Richard L; Granneman, James G; Lee, Menq-Jer

    2014-11-14

    Adipocyte lipolysis can increase the production of inflammatory cytokines such as interleukin-6 (IL-6) that promote insulin resistance. However, the mechanisms that link lipolysis with inflammation remain elusive. Acute activation of β3-adrenergic receptors (ADRB3) triggers lipolysis and up-regulates production of IL-6 in adipocytes, and both of these effects are blocked by pharmacological inhibition of hormone-sensitive lipase. We report that stimulation of ADRB3 induces expression of sphingosine kinase 1 (SphK1) and increases sphingosine 1-phosphate production in adipocytes in a manner that also depends on hormone-sensitive lipase activity. Mechanistically, we found that adipose lipolysis-induced SphK1 up-regulation is mediated by the c-Jun N-terminal kinase (JNK)/activating protein-1 signaling pathway. Inhibition of SphK1 by sphingosine kinase inhibitor 2 diminished the ADRB3-induced IL-6 production both in vitro and in vivo. Induction of IL-6 by ADRB3 activation was suppressed by siRNA knockdown of Sphk1 in cultured adipocytes and was severely attenuated in Sphk1 null mice. Conversely, ectopic expression of SphK1 increased IL-6 expression in adipocytes. Collectively, these data demonstrate that SphK1 is a critical mediator in lipolysis-triggered inflammation in adipocytes. PMID:25253697

  3. Pyrrolopyridine inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2).

    Science.gov (United States)

    Anderson, David R; Meyers, Marvin J; Vernier, William F; Mahoney, Matthew W; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Schindler, John F; Reitz, David B; Mourey, Robert J

    2007-05-31

    A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.

  4. Measuring MAP kinase activity in immune complex assays.

    Science.gov (United States)

    Cherkasova, Vera A

    2006-11-01

    I present an overview of published methods for measuring mitogen activated protein (MAP) kinase activity on endogenous associated substrates, exogenously added substrates as well as determination of activation loop phosphorylation as a read-out of kinase activity in vivo. Detailed procedures for these assays are given for two MAP kinases (MAPKs) Fus3 and Kss1 and compared with other published protocols, including the protocols for Hog1 and Mpk1 MAPKs. Measuring kinase activity in immune complex assays can serve as an approach for identification of potential substrates of protein kinases as well as for detecting other kinase-associated proteins. PMID:16890454

  5. N-Terminal Fatty Acid Substitution Increases the Leishmanicidal Activity of CA(1-7)M(2-9), a Cecropin-Melittin Hybrid Peptide

    Science.gov (United States)

    Chicharro, Cristina; Granata, Cesare; Lozano, Rosario; Andreu, David; Rivas, Luis

    2001-01-01

    In order to improve the leishmanicidal activity of the synthetic cecropin A-melittin hybrid peptide CA(1-7)M(2-9) (KWKLFKKIGAVLKVL-NH2), a systematic study of its acylation with saturated linear fatty acids was carried out. Acylation of the Nɛ-7 lysine residue led to a drastic decrease in leishmanicidal activity, whereas acylation at lysine 1, in either the α or the ɛ NH2 group, increased up to 3 times the activity of the peptide against promastigotes and increased up to 15 times the activity of the peptide against amastigotes. Leishmanicidal activity increased with the length of the fatty acid chain, reaching a maximum for the lauroyl analogue (12 carbons). According to the fast kinetics, dissipation of membrane potential, and parasite membrane permeability to the nucleic acid binding probe SYTOX green, the lethal mechanism was directly related to plasma membrane permeabilization. PMID:11502512

  6. The involvement of c-Jun N-terminal kinase signaling pathway in the protective effect of estrogen on attenuating ischemia reperfusion injury in a rat flap model%c-Jun氨基末端激酶通路参与雌激素对大鼠皮瓣缺血再灌注损伤的保护作用

    Institute of Scientific and Technical Information of China (English)

    李志敏; 巨积辉; 刘跃飞; 金乾衡; 吴建龙; 侯瑞兴

    2016-01-01

    Objeetive To observe the protective effect of estrogen against ischemia reperfusion injury in axial flaps,and investigate the role of the c-Jun N-terminal kinase signaling pathway in estrogen's protective effect.Methods An ischemia reperfusion injury model in the abdominal flap was created in 40 Wistar rats that were randomly divided into 4 groups:control (group A),ischemia reperfusion injury (group B),estrogen (group C) and JUN inhibitor (group D).Seven days postoperatively,gross observation of the flap,measurement of flap survival area and calculation of flap survival rate were carried out.The flap tissues were harvested for hematoxylin-eosin staining to observe histological changes,and for Westem blot to quantify JNK,p-JNK and MKP-5 expression.The relationship between flap survival and JNK expression was analyzed.Results Flaps in groups C and D grew well.Flap survival rates in these two groups were significantly higher than that in group B,while pathological changes were milder.Expressions of JNK and p-JNK were significantly lower in flaps of groups C and D than in flaps of group B,while expression of JNK negative regulator MKP-5 was the opposite.Conclusion Estrogen can significantly improve the ischemia reperfusion injury in flaps and increase flap survival rate.The potential mechanism of estrogen's protective effect can be through regulating Jun N-terminal kinase signaling pathway.%目的 观察雌激素对皮瓣缺血再灌注损伤的保护作用,初步研究c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)通路与雌激素保护作用的相关性.方法 取40只Wistar大鼠建立大鼠腹部皮瓣缺血再灌注损伤模型,随机分为健康对照组(A组)、缺血再灌注损伤组(B组)、雌激素组(C组)、JNK抑制剂组(D组).术后第七天观察各组皮瓣大体情况,测量皮瓣成活面积并计算成活率,HE染色观察各组皮瓣组织学改变,测定皮瓣组织中JNK、p-JNK、丝裂原活化蛋白激酶磷酸酶-5(MKP-5)的表达.

  7. The dark and bright sides of an enzyme: a three dimensional structure of the N-terminal domain of Zophobas morio luciferase-like enzyme, inferences on the biological function and origin of oxygenase/luciferase activity.

    Science.gov (United States)

    Prado, R A; Santos, C R; Kato, D I; Murakami, M T; Viviani, V R

    2016-05-11

    Beetle luciferases, the enzymes responsible for bioluminescence, are special cases of CoA-ligases which have acquired a novel oxygenase activity, offering elegant models to investigate the structural origin of novel catalytic functions in enzymes. What the original function of their ancestors was, and how the new oxygenase function emerged leading to bioluminescence remains unclear. To address these questions, we solved the crystal structure of a recently cloned Malpighian luciferase-like enzyme of unknown function from Zophobas morio mealworms, which displays weak luminescence with ATP and the xenobiotic firefly d-luciferin. The three dimensional structure of the N-terminal domain showed the expected general fold of CoA-ligases, with a unique carboxylic substrate binding pocket, permitting the binding and CoA-thioesterification activity with a broad range of carboxylic substrates, including short-, medium-chain and aromatic acids, indicating a generalist function consistent with a xenobiotic-ligase. The thioesterification activity with l-luciferin, but not with the d-enantiomer, confirms that the oxygenase activity emerged from a stereoselective impediment of the thioesterification reaction with the latter, favoring the alternative chemiluminescence oxidative reaction. The structure and site-directed mutagenesis support the involvement of the main-chain amide carbonyl of the invariant glycine G323 as the catalytic base for luciferin C4 proton abstraction during the oxygenase activity in this enzyme and in beetle luciferases (G343). PMID:27101527

  8. Janus Activated Kinase inhibition in Myelofibrosis

    Directory of Open Access Journals (Sweden)

    H Malhotra

    2012-01-01

    Full Text Available Janus Activated Kinase (JAK 2 plays an important role in the pathogenesis of myelofibrosis (MF. Ruxolitinib (INCB018424, Jakafi is a potent dual JAK1 and JAK2 inhibitor. In November 2011, it became approved by the US FDA for the treatment of intermediate or high-risk MF. This review shall outline the role of Ruxolitinib in the current management of MF and its potential future.

  9. 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

    International Nuclear Information System (INIS)

    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 μg/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were 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.

  10. JNK通路在新生鼠坏死性小肠结肠炎模型中的表达及其意义%Expression and Significance of c-Jun N-terminal Kinase in Necrotizing Enterocolitis of Neonatal Rats

    Institute of Scientific and Technical Information of China (English)

    习隽丽; 刘先州

    2010-01-01

    目的 观察新生大鼠坏死性小肠结肠炎(necrotizing enterocolitis, NEC)中c-Jun氨基末端激酶(c-Jun N-terminal Kinase, JNK)的激化及胞内分布规律,探讨其在NEC发病机制中的作用.方法 新生鼠随机分为正常对照组和NEC动物模型组,每组10只,于母鼠喂养3d,第4d处死.取回盲部肠组织1~2cm,用10%的甲醛立即固定,常规石蜡包埋,分别作组织形态学检查和免疫组织化学检测JNK的表达.结果 新生大鼠NEC模型中JNK在肠道细胞的胞浆中黄棕色染色颗粒表达并伴有向核转移,其阳性表达明显高于正常对照组.结论 JNK信号通路可能参与了NEC发病过程,并起着信号传导作用.

  11. Creatine kinase activity is associated with blood pressure

    NARCIS (Netherlands)

    L.M. Brewster; G. Mairuhu; N.R. Bindraban; R.P. Koopmans; J.F. Clark; G.A. van Montfrans

    2006-01-01

    Background - We previously hypothesized that high activity of creatine kinase, the central regulatory enzyme of energy metabolism, facilitates the development of high blood pressure. Creatine kinase rapidly provides adenosine triphosphate to highly energy-demanding processes, including cardiovascula

  12. Long-term effects of peroxisome proliferator-activated receptor ligand bezafibrate on N-terminal pro-B type natriuretic peptide in patients with advanced functional capacity impairment

    Directory of Open Access Journals (Sweden)

    Matas Zipora

    2009-01-01

    Full Text Available Abstract Background The effects of pan-peroxisome proliferator-activated receptor (PPAR ligand bezafibrate on N-terminal pro-B type natriuretic peptide (ProBNP level in patients with coronary artery disease (CAD is unknown. The current study aimed to investigate the long-term effects of bezafibrate on ProBNP level in patients with pre-existing CAD and advanced functional capacity impairment. Methods Metabolic and inflammatory parameters were analyzed from stored frozen serum samples obtained from 108 patients enrolled in the Bezafibrate Infarction Prevention (BIP Study. They presented with New York Heart Association (NYHA functional class III, comprising 58 patients in the bezafibrate group and 50 in the placebo groups, and completed a 2-year prospective, double-blind, placebo-controlled follow-up. Results During follow-up ProBNP level did not change significantly in the placebo group, whereas it increased slightly in the bezafibrate group, which was older and with lower baseline ProBNP values. No significant differences between the groups were found for ProBNP levels after 2 year of follow-up. Analysis-of-covariance (ANCOVA -taking into account age and baseline ProBNP level- showed that bezafibrate was not associated with longitudinal ProBNP changes during the follow-up period (p = 0.3. Conclusion Long-term treatment by bezafibrate was not associated with longitudinal ProBNP changes in patients with pre-existing CAD and advanced functional capacity impairment.

  13. Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase

    Science.gov (United States)

    Shimizu, M.; Wang, W.; Walch, E. T.; Dunne, P. W.; Epstein, H. F.

    2000-01-01

    Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.

  14. 神经干细胞对老化小胶质细胞存活及JNK信号通路的调控作用%Regulation of neural stem cells on viability and c-Jun N-terminal kinase signaling of aging microglia cells

    Institute of Scientific and Technical Information of China (English)

    武爱梅; 赵江明; 吴蕾; 方辉; 王宇; 吴惠梅

    2013-01-01

    Objective To investigate the regulation of neural stem cells (NSCs) on the viability and stress-activated kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling of aging microglia cells.Methods Primary microglia cells were isolated from 12-18 months old ICR mouse and NSCs were isolated from 12.5 days pregnancy ICR mouse.Staining test with Isolectin-B4,the specific marker for microglia,was performed and NSCs were verified by expression of nestin with immunofluorescence.Four groups were chosen in our experiment,including microglia cells group,co-cultured group,Sp600125 stimulated group and Sp600125-stimulation co-cultured group; in the Sp600125 stimulated group,microglia cells were pretreated with 20 ng/mL SP600125,a specific inhibitor of JNK,for four h; in the co-cultured group,microglia and NSC cells (1:4) were co-cultured using a Millicell Hanging Cell Culture Insert plates; and Sp600125-stimulation co-cultured group was also pretreated with 20 ng/mL SP600125 for four h firstly,and then,NSCs were added to co-culture with microglia cells for 3 d.MTT assay was performed to analyze the proliferation ability; Western blotting was used to detect the protein expression level of phosphorylated JNK signaling.Results After culturing for 2 weeks,primary microglia cells had a good adherence ability and strong refractivity.Staining test with Isolectin-B4 showed that the purity reached 80%.Neural stem cells grew like suspended spheres and nestin-positive.As compared with microglia cells group and stem cells group,co-cultured group had a significantly higher proliferation ability in MTT assay (P<0.05).The phosphorylated JNK level in the co-cultured group was significantly up-regulated as compared with that in the microglia cells group (P<0.05);Sp600125-stimulation co-cultured group had obviously higher phosphorylated JNK level than that in the Sp600125-stimulated group (P<0.05).Conclusion NSCs might promote the survival of aging microglia cells through activation of JNK

  15. Protein kinase D activity controls endothelial nitric oxide synthesis

    OpenAIRE

    Aicart-Ramos, Clara; Sánchez-Ruiloba, Lucía; Gómez-Parrizas, Mónica; Zaragoza, Carlos; Iglesias, Teresa; Rodríguez-Crespo, Ignacio

    2014-01-01

    Vascular endothelial growth factor (VEGF) regulates key functions of the endothelium, such as angiogenesis or vessel repair in processes involving endothelial nitric oxide synthase (eNOS) activation. One of the effector kinases that become activated in endothelial cells upon VEGF treatment is protein kinase D (PKD). Here, we show that PKD phosphorylates eNOS, leading to its activation and a concomitant increase in NO synthesis. Using mass spectrometry, we show that the purified active kinase ...

  16. A Molecular Brake in the Kinase Hinge Region Regulates the Activity of Receptor Tyrosine Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Chen,H.; Ma, J.; Li, W.; Eliseenkova, A.; Xu, C.; Neubert, T.; Miller, W.; Mohammadi, M.

    2007-01-01

    Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory 'molecular brake' mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.

  17. Association and regulation of casein kinase 2 activity by adenomatous polyposis coli protein

    Science.gov (United States)

    Homma, Miwako Kato; Li, Dongxia; Krebs, Edwin G.; Yuasa, Yasuhito; Homma, Yoshimi

    2002-01-01

    Mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis coli and also sporadic colorectal cancer development. By using antibodies raised against the N-terminal region of APC protein, we have detected the variable masses of endogenous APC proteins in individual cell lines established from human colorectal carcinomas caused by nonsense mutations of the gene. Phosphorylation of immunoprecipitates of full-length and truncated APC were observed in in vitro kinase reaction, indicating association of APC with protein kinase activity. The kinase activity complexed with APC was sensitive to heparin and used GTP as phosphoryl donor, suggesting an involvement of casein kinase 2 (CK2). Both CK2α- and β-subunits were found to associate with APC in immunoprecipitates as well as in pull-down assays, with preferential interaction of APC with tetrameric CK2 holoenzyme. In synchronized cell populations, the association of APC with CK2 was cell cycle dependent, with the highest association in G2/M. Unexpectedly, APC immunoprecipitates containing full-length APC protein inhibited CK2 in vitro, whereas immunoprecipitates of truncated APC had little effect. This was confirmed by using recombinant APC, and the inhibitory region was localized to the C terminus of APC between residues 2086 and 2394. Overexpression of this fragment in SW480 cells suppressed cell proliferation rates as well as tumorigenesis. These results demonstrate a previously uncharacterized functional interaction between the tumor suppressor protein APC and CK2 and suggest that growth-inhibitory effects of APC may be regulated by inhibition of CK2. PMID:11972058

  18. Antiestrogenic activity of flavnoid phytochemicals mediated via c-Jun N-terminal protein kinase pathway. Cell-type specific regulation of estrogen receptor alpha

    Science.gov (United States)

    Flavonoid phytochemicals act as both agonists and antagonists of the human estrogen receptors (ERs). While a number of these compounds act by directly binding to the ER, certain phytochemicals, such as the flavonoid compounds chalcone and flavone, elicit antagonistic effects on estrogen signaling in...

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

    Science.gov (United States)

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

    2015-11-15

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

  20. High quality, small molecule-activity datasets for kinase research.

    Science.gov (United States)

    Sharma, Rajan; Schürer, Stephan C; Muskal, Steven M

    2016-01-01

    Kinases regulate cell growth, movement, and death. Deregulated kinase activity is a frequent cause of disease. The therapeutic potential of kinase inhibitors has led to large amounts of published structure activity relationship (SAR) data. Bioactivity databases such as the Kinase Knowledgebase (KKB), WOMBAT, GOSTAR, and ChEMBL provide researchers with quantitative data characterizing the activity of compounds across many biological assays. The KKB, for example, contains over 1.8M kinase structure-activity data points reported in peer-reviewed journals and patents. In the spirit of fostering methods development and validation worldwide, we have extracted and have made available from the KKB 258K structure activity data points and 76K associated unique chemical structures across eight kinase targets. These data are freely available for download within this data note. PMID:27429748

  1. Differential AMP-activated Protein Kinase (AMPK) Recognition Mechanism of Ca2+/Calmodulin-dependent Protein Kinase Kinase Isoforms.

    Science.gov (United States)

    Fujiwara, Yuya; Kawaguchi, Yoshinori; Fujimoto, Tomohito; Kanayama, Naoki; Magari, Masaki; Tokumitsu, Hiroshi

    2016-06-24

    Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKKβ phosphorylates Thr(172) in the AMPKα subunit more efficiently than CaMKKα, with a lower Km (∼2 μm) for AMPK, whereas the CaMKIα phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKKα/CaMKKβ chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKKβ/Ile(322) in CaMKKα confer, at least in part, a distinct recognition of AMPK but not of CaMKIα. PMID:27151216

  2. Fatal infantile cardiac glycogenosis with phosphorylase kinase deficiency and a mutation in the gamma2-subunit of AMP-activated protein kinase.

    Science.gov (United States)

    Akman, Hasan O; Sampayo, James N; Ross, Fiona A; Scott, John W; Wilson, Gregory; Benson, Lee; Bruno, Claudio; Shanske, Sara; Hardie, D Grahame; Dimauro, Salvatore

    2007-10-01

    A 10-wk-old infant girl with severe hypertrophy of the septal and atrial walls by cardiac ultrasound, developed progressive ventricular wall thickening and died of aspiration pneumonia at 5 mo of age. Postmortem examination revealed ventricular hypertrophy and massive atrial wall thickening due to glycogen accumulation. A skeletal muscle biopsy showed increased free glycogen and decreased activity of phosphorylase b kinase (PHK). The report of a pathogenic mutation (R531Q) in the gene (PRKAG2) encoding the gamma2 subunit of AMP-activated protein kinase (AMPK) in three infants with congenital hypertrophic cardiomyopathy, glycogen storage, and "pseudo PHK deficiency" prompted us to screen this gene in our patient. We found a novel (R384T) heterozygous mutation in PRKAG2, affecting an arginine residue in the N-terminal AMP-binding domain. Like R531Q, this mutation reduces the binding of AMP and ATP to the isolated nucleotide-binding domains, and prevents activation of the heterotrimer by metabolic stress in intact cells. The mutation was not found in DNA from the patient's father, the only available parent, and is likely to have arisen de novo. Our studies confirm that mutations in PRKAG2 can cause fatal infantile cardiomyopathy, often associated with apparent PHK deficiency.

  3. [Protein kinase C activation induces platelet apoptosis].

    Science.gov (United States)

    Zhao, Li-Li; Chen, Meng-Xing; Zhang, Ming-Yi; Dai, Ke-Sheng

    2013-10-01

    Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelets, or in the development of some platelet-related diseases. However, up to now, a little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase C (PKC) is highly expressed in platelets and plays central roles in regulating platelet functions. Although there is evidence indicating that PKC is involved in the regulation of apoptosis of nucleated cells, it is still unclear whether PKC plays a role in platelet apoptosis. The aim of this study was to investigate the role of PKC in platelet apoptosis. The effects of PKC on mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) exposure, and caspase-3 activation of platelets were analyzed by flow cytometry and Western blot. The results showed that the ΔΨm depolarization in platelets was induced by PKC activator in time-dependent manner, and the caspase-3 activation in platelets was induced by PKC in concentration-dependent manner. However, the platelets incubated with PKC inhibitor did not results in ΔΨm depolarization and PS exposure. It is concluded that the PKC activation induces platelet apoptosis through influencing the mitochondrial functions and activating caspase 3. The finds suggest a novel mechanism for PKC in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.

  4. Antithrombin up-regulates AMP-activated protein kinase signalling during myocardial ischaemia/reperfusion injury.

    Science.gov (United States)

    Ma, Yina; Wang, Jinli; Gao, Junjie; Yang, Hui; Wang, Yanqing; Manithody, Chandrashekhara; Li, Ji; Rezaie, Alireza R

    2015-02-01

    Antithrombin (AT) is a protein of the serpin superfamily involved in regulation of the proteolytic activity of the serine proteases of the coagulation system. AT is known to exhibit anti-inflammatory and cardioprotective properties when it binds to heparan sulfate proteoglycans (HSPGs) on vascular cells. AMP-activated protein kinase (AMPK) plays an important cardioprotective role during myocardial ischaemia and reperfusion (I/R). To determine whether the cardioprotective signaling function of AT is mediated through the AMPK pathway, we evaluated the cardioprotective activities of wild-type AT and its two derivatives, one having high affinity and the other no affinity for heparin, in an acute I/R injury model in C57BL/6J mice in which the left anterior descending coronary artery was occluded. The serpin derivatives were given 5 minutes before reperfusion. The results showed that AT-WT can activate AMPK in both in vivo and ex vivo conditions. Blocking AMPK activity abolished the cardioprotective function of AT against I/R injury. The AT derivative having high affinity for heparin was more effective in activating AMPK and in limiting infraction, but the derivative lacking affinity for heparin was inactive in eliciting AMPK-dependent cardioprotective activity. Activation of AMPK by AT inhibited the inflammatory c-Jun N-terminal protein kinase (JNK) pathway during I/R. Further studies revealed that the AMPK activity induced by AT also modulates cardiac substrate metabolism by increasing glucose oxidation but inhibiting fatty acid oxidation during I/R. These results suggest that AT binds to HSPGs on heart tissues to invoke a cardioprotective function by triggering cardiac AMPK activation, thereby attenuating JNK inflammatory signalling pathways and modulating substrate metabolism during I/R. PMID:25230600

  5. [Determination of riboflavin kinase activity in yeast].

    Science.gov (United States)

    Shavlovsky, G M; Kashchenko, V E

    1975-01-01

    It is established that the main reason of the riboflavin kinase (RFK, EC 2.7.1.26) low specific activity in the cell-free extracts of the yeast Pichia guillermondii Wickerham ATCC 9058 is the presence of alkaline phosphatase (EC 3.1.3.1), effectively destructing flaven mononucleotide. By chromatography of the cell-free extracts of P. guillermondii on DEAE-Sephadex A-50, CM-Sphadex C-50, CM-cellulose, Sephadexes G-75 and G-100 RFK and alkaline phosphatase may be separated completely. Any of these procedures results in a several times increase of the RFK activity as compared with the initial preparation. One failed to obtain a similar effect by fractionation of the extracts with amminium sulphate and by hydroxylapatite chromatography. A simple method is developed for determining the activity of RFK in the cell-free extracts of yeast on the basis of negative adsorption of this enzyme on DEAE-Sephadex A-50. A selective inhibition of alkaline phosphatase by ions Be2+ and F- yields a less satisfactory result. The data are presented on the PFK activity of certain species of flavinogenic (Pichia guillermondii, Torulopsis camdida) and non-flavinogenic (Pichia ohmeri, Candida utilis, Saccharomyces cervisiae) yeast. PMID:174262

  6. Discovery and Characterization of a Cell-Permeable, Small-Molecule c-Abl Kinase Activator that Binds to the Myristoyl Binding Site

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Jingsong; Campobasso, Nino; Biju, Mangatt P.; Fisher, Kelly; Pan, Xiao-Qing; Cottom, Josh; Galbraith, Sarah; Ho, Thau; Zhang, Hong; Hong, Xuan; Ward, Paris; Hofmann, Glenn; Siegfried, Brett; Zappacosta, Francesca; Washio, Yoshiaki; Cao, Ping; Qu, Junya; Bertrand, Sophie; Wang, Da-Yuan; Head, Martha S.; Li, Hu; Moores, Sheri; Lai, Zhihong; Johanson, Kyung; Burton, George; Erickson-Miller, Connie; Simpson, Graham; Tummino, Peter; Copeland, Robert A.; Oliff, Allen (GSKPA)

    2014-10-02

    c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the {alpha}I helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the {alpha}I helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.

  7. Activation of Phosphorylase Kinase by Physiological Temperature.

    Science.gov (United States)

    Herrera, Julio E; Thompson, Jackie A; Rimmer, Mary Ashley; Nadeau, Owen W; Carlson, Gerald M

    2015-12-29

    In the six decades since its discovery, phosphorylase kinase (PhK) from rabbit skeletal muscle has usually been studied at 30 °C; in fact, not a single study has examined functions of PhK at a rabbit's body temperature, which is nearly 10 °C greater. Thus, we have examined aspects of the activity, regulation, and structure of PhK at temperatures between 0 and 40 °C. Between 0 and 30 °C, the activity at pH 6.8 of nonphosphorylated PhK predictably increased; however, between 30 and 40 °C, there was a dramatic jump in its activity, resulting in the nonactivated enzyme having a far greater activity at body temperature than was previously realized. This anomalous change in properties between 30 and 40 °C was observed for multiple functions, and both stimulation (by ADP and phosphorylation) and inhibition (by orthophosphate) were considerably less pronounced at 40 °C than at 30 °C. In general, the allosteric control of PhK's activity is definitely more subtle at body temperature. Changes in behavior related to activity at 40 °C and its control can be explained by the near disappearance of hysteresis at physiological temperature. In important ways, the picture of PhK that has emerged from six decades of study at temperatures of ≤30 °C does not coincide with that of the enzyme studied at physiological temperature. The probable underlying mechanism for the dramatic increase in PhK's activity between 30 and 40 °C is an abrupt change in the conformations of the regulatory β and catalytic γ subunits between these two temperatures.

  8. The autophosphorylation and p34cdc2 phosphorylation sites of casein kinase-2 beta-subunit are not essential for reconstituting the fully-active heterotetrameric holoenzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Issinger, O G;

    1993-01-01

    Two mutants of human casein kinase-2 beta-subunit with short deletions at either their amino (delta 1-4) or carboxy (delta 209-215) terminal side have been created that have lost the capability to undergo autophosphorylation and p34cdc2 mediated phosphorylation, respectively. Both mutants give rise...... to reconstituted CK2 holoenzymes displaying basal catalytic activity, thermostability and responsiveness to polylysine, identical to those of wild-type holoenzyme, whose reconstitution, moreover, is not affected by previous phosphorylation of the beta-subunit at either its N-terminal or C-terminal sites. Unlike...

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

    Science.gov (United States)

    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.

  10. Multiple host kinases contribute to Akt activation during Salmonella infection.

    Directory of Open Access Journals (Sweden)

    Bernhard Roppenser

    Full Text Available SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4 P2/PI(3-5 P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4 P2/PI(3-5 P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

  11. c-Jun氨基末端激酶1反义真核表达载体及其蛋白缺陷细胞株的构建与鉴定%Construction and identification of antisense c-Jun N-terminal kinase 1 eukaryotic fluorescent expressing plasmids and JNK1+ human embryo lung fibroblasts cell line

    Institute of Scientific and Technical Information of China (English)

    徐辉; 何晓庆; 陈瑞; 尹仕伟; 彭雷; 王国强; 李爱萍; 周建伟; 刘起展

    2008-01-01

    目的 构建反义JNK1荧光真核细胞表达载体,建立JNK1蛋白缺陷人胚肺成纤维细胞(HELF)株.方法 用Trizol试剂抽提HELF细胞中总RNA,以反转录PCR扩增JNK1目的 片断,双酶切,纯化PCR产物后,反向插入pEGFP-C1绿色荧光质粒,构建反义pEGFP-C1-asJNK1真核表达载体;大量抽提质粒并转染至HELF细胞中.24 h后使用G418筛选,挑选单克隆细胞扩大培养,经荧光显微成像和蛋白免疫印迹鉴定.结果 pEGFP-C1-asJNK1表达载体DNA测序结果与预期目的 片断序列一致,且JNK1蛋白表达水平明显抑制.结论 反义pEGFP-C1-asJNK1真核表达载体构建成功,JNK1蛋白质缺陷HELF细胞株成功建立.%Objective To construct antisense c-Jun N-terminal kinase 1 (JNK1) eukaryotic fluorescent expressing vector and JNK1+ human embryo lung fibroblasts cell line. Methods Trizol reagent was used to extract total RNA in HELF. The proper primers of JNK1 were chosen and synthesized. RT-PCR and gene recombinant techniques were used to construct the fragment of JNK1. After purification, the PCR products were cut, and JNK1 were inserted reversely into eukaryotic fluorescent expressing vector pEGFP-C1. Enzyme-cutting and DNA auto-sequencing were used to prove the successful construction of JNK1 eukaryotic expressing vector. Then plasmids were extracted and transfected into HELF cells and screen by G418 24 h later. Monoclone was chosen and cultured. Fluorescent imaging and Western blot were used to identify the JNK+HELF cell line. Results Sequence analysis of pEGFP-C1-as JNK1 plasmids was same as expected. The expression level of JNK1 was inhibited markedly. Conclusion Construction of antisensc JNK1 eukaryotic fluorescent expressing vectors and JNK + HELF cell line is successful.

  12. Rat mammary carcinogenesis induced by in situ expression of constitutive Raf kinase activity is prevented by tethering Raf to the plasma membrane.

    Science.gov (United States)

    McFarlin, Daniel R; Gould, Michael N

    2003-06-01

    Mammary carcinogenesis induced through expression of activated Raf was investigated using a model in which retroviral vectors were infused into the central ducts of rat mammary glands. This model allows efficient expression of experimental proteins in a small fraction of endogenous mammary epithelial cells in situ. We previously reported that Raf is the dominant oncogenic signaling pathway from activated Ras in rat mammary glands. We show here that mammary gland carcinogenesis is rapidly induced by the expression of c-Raf-1 kinase that is activated by N-terminal truncation (Delta-Raf). Interestingly, targeting Raf to the plasma membrane via C-terminal fusion with Ras membrane localization signals (Raf-Caax) induces Raf kinase activity that transforms 3T3 cells more frequently than Delta-Raf, yet in situ expression of Raf-Caax does not induce mammary carcinomas. To investigate these contrasting results and begin elucidating the mechanisms of Raf-induced mammary carcinogenesis, we combined both activating mutations (N-terminal truncation and C-terminal membrane localization motifs) in one Raf construct (Delta-Raf-Caax). While Delta-Raf-Caax transforms 3T3 cells more efficiently than Delta-Raf or Raf-Caax, in situ expression of Delta-Raf-Caax does not induce carcinomas in vivo, demonstrating that lipid modification on the C-terminus of Delta-Raf negates its oncogenic potential in rat mammary gland.

  13. Asymmetric Tyrosine Kinase Arrangements in Activation or Autophosphorylation of Receptor Tyrosine Kinases

    Energy Technology Data Exchange (ETDEWEB)

    J Bae; J Schlessinger

    2011-12-31

    Receptor tyrosine kinases (RTKs) play important roles in the control of many cellular processes including cell proliferation, cell adhesion, angiogenesis, and apoptosis. Ligand-induced dimerization of RTKs leads to autophosphorylation and activation of RTKs. Structural studies have shown that while isolated ectodomains of several RTKs form symmetric dimers the isolated cytoplasmic kinase domains of epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) form asymmetric dimers during their activation. Binding of one kinase molecule of EGFR to a second kinase molecule asymmetrically leads to stimulation of kinase activity and enhanced autophosphorylation. Furthermore, the structures of the kinase domain of FGFR1 and FGFR2 reveal the formation of asymmetric interfaces in the processes of autophosphorylation at their specific phosphotyrosine (pY) sites. Disruption of asymmetric dimer interface of EGFR leads to reduction in enzymatic activity and drastic reduction of autophosphorylation of FGFRs in ligandstimulated live cells. These studies demonstrate that asymmetric dimer formation is as a common phenomenon critical for activation and autophosphorylation of RTKs.

  14. AcAP5蛋白N端片段的表达、纯化与活性评价%Expression, purification and evaluation of N-terminal domain of AcAP5 with Factor Xa inhibitory activity

    Institute of Scientific and Technical Information of China (English)

    刘爱华; 朱元军; 刘晓岩; 王银叶

    2013-01-01

    Ancylostoma anticoagulant peptide 5 (AcAP5) is a strong inhibitor of human coagulation factor Xa (FXa).The N-terminal residues (N40) of AcAP5 contains a domain that could combine with FXa.In order to determine whether N40 protein has FXa inhibitory effect,we cloned,expressed and purified the protein for activity evaluation.The DNA fragment coding N40 was amplified by PCR,cloned into pET-30a to construct recombinant plasmid pET30a-N40,and subsequently transformed into E.coli.BL21 (DE3).Expression of N40 was induced by isopropyl β-D-l-thiogalactopyranoside (IPTG),and the interest protein was identified by SDS-PAGE and purified using one-step nickel (Ni) affinity chromatography.Under the optimal expression condition (0.05 mM IPTG for 6 h at 37 ℃),the purity of N40 reached 90%.We also evaluated the inhibition activity of N40 protein on FXa,finding the IC50 was 4.58× 10 5 mol/L.This study suggests the N40 of AcAP5 could combine with FXa to inhibit FXa activity.%犬钩虫抗凝肽5 (Ancylostoma anticoagulant peptide 5,AcAP5)N端40个氨基酸片段(N40)含有与FXa结合的结构域.为研究N40的FXa抑制作用,我们克隆、表达和纯化了N40,并检测其生物活性.用PCR扩增N40基因;将PCR产物克隆至原核表达载体pET-30a中,构建质粒pET30a-N40;将其转化入大肠杆菌E.coli.BL21 (DE3)中,IPTG诱导目的蛋白表达,SDS-PAGE电泳鉴定,经过镍柱亲和层析纯化,用BCA法进行蛋白定量,测定该蛋白抑制FXa的活性.限制性酶切鉴定和基因测序结果显示重组质粒构建成功;SDS-PAGE结果显示目的蛋白在E.coli.BL21(DE3)中为可溶性表达,亲和层析纯化后获得了纯度约90%的蛋白,经活性鉴定该蛋白确有抑制FXa的活性.

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

    DEFF Research Database (Denmark)

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia;

    2003-01-01

    Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline......-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50% by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None...... of the inhibitors reduced adrenaline-induced HSL activation in soleus muscle. Both phorbol-12-myristate-13-acetate (PMA), which activates PKC and, in turn, ERK, and caffeine, which increases intracellular Ca2+ without eliciting contraction, increased HSL activity. Activated ERK increased HSL activity in supernatant...

  16. Inhibition of human insulin gene transcription and MafA transcriptional activity by the dual leucine zipper kinase.

    Science.gov (United States)

    Stahnke, Marie-Jeannette; Dickel, Corinna; Schröder, Sabine; Kaiser, Diana; Blume, Roland; Stein, Roland; Pouponnot, Celio; Oetjen, Elke

    2014-09-01

    Insulin biosynthesis is an essential β-cell function and inappropriate insulin secretion and biosynthesis contribute to the pathogenesis of diabetes mellitus type 2. Previous studies showed that the dual leucine zipper kinase (DLK) induces β-cell apoptosis. Since β-cell dysfunction precedes β-cell loss, in the present study the effect of DLK on insulin gene transcription was investigated in the HIT-T15 β-cell line. Downregulation of endogenous DLK increased whereas overexpression of DLK decreased human insulin gene transcription. 5'- and 3'-deletion human insulin promoter analyses resulted in the identification of a DLK responsive element that mapped to the DNA binding-site for the β-cell specific transcription factor MafA. Overexpression of DLK wild-type but not its kinase-dead mutant inhibited MafA transcriptional activity conferred by its transactivation domain. Furthermore, in the non-β-cell line JEG DLK inhibited MafA overexpression-induced human insulin promoter activity. Overexpression of MafA and DLK or its kinase-dead mutant into JEG cells revealed that DLK but not its mutant reduced MafA protein content. Inhibition of the down-stream DLK kinase c-Jun N-terminal kinase (JNK) by SP600125 attenuated DLK-induced MafA loss. Furthermore, mutation of the serine 65 to alanine, shown to confer MafA protein stability, increased MafA-dependent insulin gene transcription and prevented DLK-induced MafA loss in JEG cells. These data suggest that DLK by activating JNK triggers the phosphorylation and degradation of MafA thereby attenuating insulin gene transcription. Given the importance of MafA for β-cell function, the inhibition of DLK might preserve β-cell function and ultimately retard the development of diabetes mellitus type 2. PMID:24726898

  17. Protein Kinase Cδ mediates the activation of Protein Kinase D2 in Platelets

    OpenAIRE

    Bhavanasi, Dheeraj; Kim, Soochong; Goldfinger, Lawrence E.; Kunapuli, Satya P.

    2011-01-01

    Protein Kinase D (PKD) is a subfamily of serine/threonine specific family of kinases, comprised of PKD1, PKD2 and PKD3 (PKCμ, PKD2 and PKCν in humans). It is known that PKCs activate PKD, but the relative expression of isoforms of PKD or the specific PKC isoform/s responsible for its activation in platelets is not known. This study is aimed at investigating the pathway involved in activation of PKD in platelets. We show that PKD2 is the major isoform of PKD that is expressed in human as well ...

  18. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies.

    Directory of Open Access Journals (Sweden)

    Westa Domanova

    Full Text Available In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds, making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same

  19. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies

    Science.gov (United States)

    Chaudhuri, Rima; Yang, Pengyi; Vafaee, Fatemeh; Fazakerley, Daniel; Humphrey, Sean; James, David; Kuncic, Zdenka

    2016-01-01

    In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds), making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs) is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE) in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same linear consensus motif

  20. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies.

    Science.gov (United States)

    Domanova, Westa; Krycer, James; Chaudhuri, Rima; Yang, Pengyi; Vafaee, Fatemeh; Fazakerley, Daniel; Humphrey, Sean; James, David; Kuncic, Zdenka

    2016-01-01

    In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds), making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs) is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE) in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same linear consensus motif

  1. Local anesthetics induce apoptosis in human thyroid cancer cells through the mitogen-activated protein kinase pathway.

    Directory of Open Access Journals (Sweden)

    Yuan-Ching Chang

    Full Text Available Local anesthetics are frequently used in fine-needle aspiration of thyroid lesions and locoregional control of persistent or recurrent thyroid cancer. Recent evidence suggests that local anesthetics have a broad spectrum of effects including inhibition of cell proliferation and induction of apoptosis in neuronal and other types of cells. In this study, we demonstrated that treatment with lidocaine and bupivacaine resulted in decreased cell viability and colony formation of both 8505C and K1 cells in a dose-dependent manner. Lidocaine and bupivacaine induced apoptosis, and necrosis in high concentrations, as determined by flow cytometry. Lidocaine and bupivacaine caused disruption of mitochondrial membrane potential and release of cytochrome c, accompanied by activation of caspase 3 and 7, PARP cleavage, and induction of a higher ratio of Bax/Bcl-2. Based on microarray and pathway analysis, apoptosis is the prominent transcriptional change common to lidocaine and bupivacaine treatment. Furthermore, lidocaine and bupivacaine attenuated extracellular signal-regulated kinase 1/2 (ERK1/2 activity and induced activation of p38 mitogen-activated protein kinase (MAPK and c-jun N-terminal kinase. Pharmacological inhibitors of MAPK/ERK kinase and p38 MAPK suppressed caspase 3 activation and PARP cleavage. Taken together, our results for the first time demonstrate the cytotoxic effects of local anesthetics on thyroid cancer cells and implicate the MAPK pathways as an important mechanism. Our findings have potential clinical relevance in that the use of local anesthetics may confer previously unrecognized benefits in the management of patients with thyroid cancer.

  2. Local anesthetics induce apoptosis in human thyroid cancer cells through the mitogen-activated protein kinase pathway.

    Science.gov (United States)

    Chang, Yuan-Ching; Hsu, Yi-Chiung; Liu, Chien-Liang; Huang, Shih-Yuan; Hu, Meng-Chun; Cheng, Shih-Ping

    2014-01-01

    Local anesthetics are frequently used in fine-needle aspiration of thyroid lesions and locoregional control of persistent or recurrent thyroid cancer. Recent evidence suggests that local anesthetics have a broad spectrum of effects including inhibition of cell proliferation and induction of apoptosis in neuronal and other types of cells. In this study, we demonstrated that treatment with lidocaine and bupivacaine resulted in decreased cell viability and colony formation of both 8505C and K1 cells in a dose-dependent manner. Lidocaine and bupivacaine induced apoptosis, and necrosis in high concentrations, as determined by flow cytometry. Lidocaine and bupivacaine caused disruption of mitochondrial membrane potential and release of cytochrome c, accompanied by activation of caspase 3 and 7, PARP cleavage, and induction of a higher ratio of Bax/Bcl-2. Based on microarray and pathway analysis, apoptosis is the prominent transcriptional change common to lidocaine and bupivacaine treatment. Furthermore, lidocaine and bupivacaine attenuated extracellular signal-regulated kinase 1/2 (ERK1/2) activity and induced activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase. Pharmacological inhibitors of MAPK/ERK kinase and p38 MAPK suppressed caspase 3 activation and PARP cleavage. Taken together, our results for the first time demonstrate the cytotoxic effects of local anesthetics on thyroid cancer cells and implicate the MAPK pathways as an important mechanism. Our findings have potential clinical relevance in that the use of local anesthetics may confer previously unrecognized benefits in the management of patients with thyroid cancer. PMID:24586874

  3. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    Science.gov (United States)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  4. Regulatory Light Chain Phosphorylation and N-Terminal Extension Increase Cross-Bridge Binding and Power Output in Drosophila at In Vivo Myofilament Lattice Spacing

    OpenAIRE

    Miller, Mark S.; Farman, Gerrie P.; Braddock, Joan M.; Soto-Adames, Felipe N.; Irving, Thomas C.; Vigoreaux, Jim O.; Maughan, David W.

    2011-01-01

    The N-terminal extension and phosphorylation of the myosin regulatory light chain (RLC) independently improve Drosophila melanogaster flight performance. Here we examine the functional and structural role of the RLC in chemically skinned fibers at various thick and thin filament lattice spacings from four transgenic Drosophila lines: rescued null or control (Dmlc2+), truncated N-terminal extension (Dmlc2Δ2-46), disrupted myosin light chain kinase phosphorylation sites (Dmlc2S66A,S67A), and du...

  5. Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activity.

    Science.gov (United States)

    Luz, Simão; Kongsuphol, Patthara; Mendes, Ana Isabel; Romeiras, Francisco; Sousa, Marisa; Schreiber, Rainer; Matos, Paulo; Jordan, Peter; Mehta, Anil; Amaral, Margarida D; Kunzelmann, Karl; Farinha, Carlos M

    2011-11-01

    Previously, the pleiotropic "master kinase" casein kinase 2 (CK2) was shown to interact with CFTR, the protein responsible for cystic fibrosis (CF). Moreover, CK2 inhibition abolished CFTR conductance in cell-attached membrane patches, native epithelial ducts, and Xenopus oocytes. CFTR possesses two CK2 phosphorylation sites (S422 and T1471), with unclear impact on its processing and trafficking. Here, we investigated the effects of mutating these CK2 sites on CFTR abundance, maturation, and degradation coupled to effects on ion channel activity and surface expression. We report that CK2 inhibition significantly decreased processing of wild-type (wt) CFTR, with no effect on F508del CFTR. Eliminating phosphorylation at S422 and T1471 revealed antagonistic roles in CFTR trafficking: S422 activation versus T1471 inhibition, as evidenced by a severe trafficking defect for the T1471D mutant. Notably, mutation of Y512, a consensus sequence for the spleen tyrosine kinase (SYK) possibly acting in a CK2 context adjacent to the common CF-causing defect F508del, had a strong effect on both maturation and CFTR currents, allowing the identification of this kinase as a novel regulator of CFTR. These results reinforce the importance of CK2 and the S422 and T1471 residues for regulation of CFTR and uncover a novel regulation of CFTR by SYK, a recognized controller of inflammation.

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

    DEFF Research Database (Denmark)

    Peraldi, P; Frödin, M; Barnier, J V;

    1995-01-01

    In PC12 cells, cAMP stimulates the MAP kinase pathway by an unknown mechanism. Firstly, we examined the role of calcium ion mobilization and of protein kinase C in cAMP-stimulated MAP kinase activation. We show that cAMP stimulates p44mapk independently of these events. Secondly, we studied the r...

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

    DEFF Research Database (Denmark)

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

    2000-01-01

    Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2...... to be an allosteric mechanism. Furthermore, we demonstrate that anisomycin- and tumor necrosis factor-alpha-induced phosphorylation of p53 at Ser-392, which is important for the transcriptional activity of this growth suppressor protein, requires p38 MAP kinase and CK2 activities....... in the human cervical carcinoma HeLa cells by up to 8-fold, and this could be blocked by the p38 MAP kinase inhibitor SB203580. We show that p38alpha MAP kinase, in a phosphorylation-dependent manner, can directly interact with the alpha and beta subunits of CK2 to activate the holoenzyme through what appears...

  8. 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.

    Science.gov (United States)

    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.

  9. N-terminal amino acid sequences and some characteristics of fibrinolytic/hemorrhagic metalloproteinases purified from Bothrops jararaca venom.

    Science.gov (United States)

    Maruyama, Masugi; Sugiki, Masahiko; Anai, Keita; Yoshida, Etsuo

    2002-08-01

    We determined the N-terminal amino acid sequences of the fibrinolytic/hemorrhagic metalloproteinases (jararafibrases I, III and IV) purified from Bothrops jararaca venom. The N-terminal amino acid sequences of jararafibrase I and its degradation products were identical to those of jararhagin, another hemorrhagic metalloproteinase purified from the same snake venom. Together with enzymatic and immunological properties, we concluded that those two enzymes are identical. The N-terminal amino acid sequence of jararafibrase III was quite similar to C-type lectin isolated from Crotalus atrox, and the protein had a hemagglutinating activity on intact rat red blood cells. PMID:12165326

  10. Organ culture of the trigeminal ganglion induces enhanced expression of calcitonin gene-related peptide via activation of extracellular signal-regulated protein kinase 1/2

    DEFF Research Database (Denmark)

    Tajti, János; Kuris, Anikó; Vécsei, László;

    2011-01-01

    , phosphorylated ERK1/2 (pERK1/2), total ERK1/2 (tERK1/2), phosphorylated p38 (pp38), phosphorylated C-Jun-N-terminal protein kinase (pJNK), pro-calcitonin (pro-CT), CGRP receptor activity modifying protein 1 (RAMP1), glutamine synthetase (GS) and pro-CT were used. To explore molecular mechanisms involved......BACKGROUND AND OBJECTIVE: Clinical and experimental studies have revealed a central role of calcitonin gene-related peptide (CGRP) in primary headaches. The role of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in neuronal and glial cell expression of CGRP- immunoreactivity (-ir) in rat...

  11. Rapamycin induces mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) expression through activation of protein kinase B and mitogen-activated protein kinase kinase pathways.

    Science.gov (United States)

    Rastogi, Ruchi; Jiang, Zhongliang; Ahmad, Nisar; Rosati, Rita; Liu, Yusen; Beuret, Laurent; Monks, Robert; Charron, Jean; Birnbaum, Morris J; Samavati, Lobelia

    2013-11-22

    Mitogen-activated protein kinase phosphatase-1 (MKP-1), also known as dual specificity phosphatase-1 (DUSP-1), plays a crucial role in the deactivation of MAPKs. Several drugs with immune-suppressive properties modulate MKP-1 expression as part of their mechanism of action. We investigated the effect of mTOR inhibition through rapamycin and a dual mTOR inhibitor (AZD2014) on MKP-1 expression. Low dose rapamycin led to a rapid activation of both AKT and ERK pathways with a subsequent increase in MKP-1 expression. Rapamycin treatment led to phosphorylation of CREB, transcription factor 1 (ATF1), and ATF2, three transcription factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter. Inhibition of either the MEK/ERK or the AKT pathway attenuated rapamycin-mediated MKP-1 induction. AZD2014 did not activate AKT but activated the ERK pathway, leading to a moderate MKP-1 induction. Using bone marrow-derived macrophages (BMDMs) derived from wild-type (WT) mice or mice deficient in AKT1 and AKT2 isoforms or BMDM from targeted deficiency in MEK1 and MEK2, we show that rapamycin treatment led to an increased MKP1 expression in BMDM from WT but failed to do so in BMDMs lacking the AKT1 isoform or MEK1 and MEK2. Importantly, rapamycin pretreatment inhibited LPS-mediated p38 activation and decreased nitric oxide and IL-6 production. Our work provides a conceptual framework for the observed immune modulatory effect of mTOR inhibition.

  12. Enhanced casein kinase II activity in human tumour cell cultures

    DEFF Research Database (Denmark)

    Prowald, K; Fischer, H; Issinger, O G

    1984-01-01

    Casein kinase II (CKII) activity is enhanced as much as 2-3 fold in established and 4-5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells...... and in established cell lines was reduced to about the same basic level after treatment with heparin, a highly specific inhibitor of CKII activity. The activity of the cAMP-dependent protein kinase was virtually the same in fibroblasts and various human tumour cell lines investigated....

  13. Inactivation of a MAPK-like protein kinase and activation of a MBP kinase in germinating barley embryos

    NARCIS (Netherlands)

    Testerink, C.; Vennik, M.; Kijne, J.W.; Wang, M.; Heimovaara-Dijkstra, S.

    2000-01-01

    We provide evidence for involvement of two different 45 kDa protein kinases in rehydration and germination of barley embryos. In dry embryos, a myelin basic protein (MBP) phosphorylating kinase was detected, which could be immunoprecipitated with an anti-MAPK (mitogen-activated protein kinase) antib

  14. Cellular trafficking of the IL-1RI-associated kinase-1 requires intact kinase activity

    International Nuclear Information System (INIS)

    Upon stimulation of cells with interleukin-1 (IL-1) the IL-1 receptor type I (IL-1RI) associated kinase-1 (IRAK-1) transiently associates to and dissociates from the IL-1RI and thereafter translocates into the nucleus. Here we show that nuclear translocation of IRAK-1 depends on its kinase activity since translocation was not observed in EL-4 cells overexpressing a kinase negative IRAK-1 mutant (EL-4IRAK-1-K239S). IRAK-1 itself, an endogenous substrate with an apparent molecular weight of 24 kDa (p24), and exogenous substrates like histone and myelin basic protein are phosphorylated by nuclear located IRAK-1. Phosphorylation of p24 cannot be detected in EL-4IRAK-1-K239S cells. IL-1-dependent recruitment of IRAK-1 to the IL-1RI and subsequent phosphorylation of IRAK-1 is a prerequisite for nuclear translocation of IRAK-1. It is therefore concluded that intracellular localization of IRAK-1 depends on its kinase activity and that IRAK-1 may also function as a kinase in the nucleus as shown by a new putative endogenous substrate

  15. Cytotoxic Synergy Between Cytokines and NSAIDs Associated With Idiosyncratic Hepatotoxicity Is Driven by Mitogen-Activated Protein Kinases.

    Science.gov (United States)

    Maiuri, Ashley R; Breier, Anna B; Gora, Lukas F J; Parkins, Robert V; Ganey, Patricia E; Roth, Robert A

    2015-08-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most frequent causes of idiosyncratic, drug-induced liver injury (IDILI). Mechanisms of IDILI are unknown, but immune responses are suspected to underlie them. In animal models of IDILI, the cytokines tumor necrosis factor-alpha (TNFα) and interferon-gamma (IFNγ) are essential to the pathogenesis. Some drugs associated with IDILI interact with cytokines to kill hepatocytes in vitro, and mitogen-activated protein kinases (MAPKs) might play a role. We tested the hypothesis that caspases and MAPKs are involved in NSAID/cytokine-induced cytotoxicity. NSAIDs that are acetic acid (AA) derivatives and associated with IDILI synergized with TNFα in causing cytotoxicity in HepG2 cells, and IFNγ enhanced this interaction. NSAIDs that are propionic acid (PA) derivatives and cause IDILI that is of less clinical concern also synergized with TNFα, but IFNγ was without effect. Caspase inhibition prevented cytotoxicity from AA and PA derivative/cytokine treatment. Treatment with a representative AA or PA derivative induced activation of the MAPKs c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38. Inhibition of either JNK or ERK reduced cytotoxicity from cytokine interactions with AA derivatives. In contrast, an ERK inhibitor potentiated cytotoxicity from cytokine interactions with PA derivatives. An AA derivative but not a PA derivative enhanced IFNγ-mediated activation of STAT-1, and this enhancement was ERK-dependent. These findings raise the possibility that some IDILI reactions result from drug/cytokine synergy involving caspases and MAPKs and suggest that, even for drugs within the same pharmacologic class, synergy with cytokines occurs by different kinase signaling mechanisms.

  16. Regulation of endothelial protein C receptor shedding by cytokines is mediated through differential activation of MAP kinase signaling pathways

    International Nuclear Information System (INIS)

    The endothelial protein C receptor (EPCR) plays a pivotal role in coagulation, inflammation, cell proliferation, and cancer, but its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). In this study we examined the mechanisms involved in the regulation of EPCR shedding in human umbilical endothelial cells (HUVEC). Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), but not interferon-γ and interleukin-6, suppressed EPCR mRNA transcription and cell-associated EPCR expression in HUVEC. The release of sEPCR induced by IL-1β and TNF-α correlated with activation of p38 MAPK and c-Jun N-terminal kinase (JNK). EPCR shedding was also induced by phorbol 12-myristate 13-acetate, ionomycin, anisomycin, thiol oxidants or alkylators, thrombin, and disruptors of lipid rafts. Both basal and induced shedding of EPCR was blocked by the metalloproteinase inhibitors, TAPI-0 and GM6001, and by the reduced non-protein thiols, glutathione, dihydrolipoic acid, dithiothreitol, and N-acetyl-L-cysteine. Because other antioxidants and scavengers of reactive oxygen species failed to block the cleavage of EPCR, a direct suppression of metalloproteinase activity seems responsible for the observed effects of reduced thiols. In summary, the shedding of EPCR in HUVEC is effectively regulated by IL-1β and TNF-α, and downstream by MAP kinase signaling pathways and metalloproteinases.

  17. Regulation of endothelial protein C receptor shedding by cytokines is mediated through differential activation of MAP kinase signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Menschikowski, Mario, E-mail: Mario.Menschikowski@uniklinikum-dresden.de [Institute of Clinical Chemistry and Laboratory Medicine, Technical University of Dresden, Medical Faculty ' Carl Gustav Carus' , Fetscherstrasse 74, D-01307 Dresden (Germany); Hagelgans, Albert; Eisenhofer, Graeme; Siegert, Gabriele [Institute of Clinical Chemistry and Laboratory Medicine, Technical University of Dresden, Medical Faculty ' Carl Gustav Carus' , Fetscherstrasse 74, D-01307 Dresden (Germany)

    2009-09-10

    The endothelial protein C receptor (EPCR) plays a pivotal role in coagulation, inflammation, cell proliferation, and cancer, but its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). In this study we examined the mechanisms involved in the regulation of EPCR shedding in human umbilical endothelial cells (HUVEC). Interleukin-1{beta} (IL-1{beta}) and tumor necrosis factor-{alpha} (TNF-{alpha}), but not interferon-{gamma} and interleukin-6, suppressed EPCR mRNA transcription and cell-associated EPCR expression in HUVEC. The release of sEPCR induced by IL-1{beta} and TNF-{alpha} correlated with activation of p38 MAPK and c-Jun N-terminal kinase (JNK). EPCR shedding was also induced by phorbol 12-myristate 13-acetate, ionomycin, anisomycin, thiol oxidants or alkylators, thrombin, and disruptors of lipid rafts. Both basal and induced shedding of EPCR was blocked by the metalloproteinase inhibitors, TAPI-0 and GM6001, and by the reduced non-protein thiols, glutathione, dihydrolipoic acid, dithiothreitol, and N-acetyl-L-cysteine. Because other antioxidants and scavengers of reactive oxygen species failed to block the cleavage of EPCR, a direct suppression of metalloproteinase activity seems responsible for the observed effects of reduced thiols. In summary, the shedding of EPCR in HUVEC is effectively regulated by IL-1{beta} and TNF-{alpha}, and downstream by MAP kinase signaling pathways and metalloproteinases.

  18. Conservation, variability and the modeling of active protein kinases.

    Directory of Open Access Journals (Sweden)

    James D R Knight

    Full Text Available The human proteome is rich with protein kinases, and this richness has made the kinase of crucial importance in initiating and maintaining cell behavior. Elucidating cell signaling networks and manipulating their components to understand and alter behavior require well designed inhibitors. These inhibitors are needed in culture to cause and study network perturbations, and the same compounds can be used as drugs to treat disease. Understanding the structural biology of protein kinases in detail, including their commonalities, differences and modes of substrate interaction, is necessary for designing high quality inhibitors that will be of true use for cell biology and disease therapy. To this end, we here report on a structural analysis of all available active-conformation protein kinases, discussing residue conservation, the novel features of such conservation, unique properties of atypical kinases and variability in the context of substrate binding. We also demonstrate how this information can be used for structure prediction. Our findings will be of use not only in understanding protein kinase function and evolution, but they highlight the flaws inherent in kinase drug design as commonly practiced and dictate an appropriate strategy for the sophisticated design of specific inhibitors for use in the laboratory and disease therapy.

  19. Design, Synthesis and Inhibitory Activity of Photoswitchable RET Kinase Inhibitors

    Science.gov (United States)

    Ferreira, Rubén; Nilsson, Jesper R.; Solano, Carlos; Andréasson, Joakim; Grøtli, Morten

    2015-05-01

    REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase required for normal development and maintenance of neurons of the central and peripheral nervous systems. Deregulation of RET and hyperactivity of the RET kinase is intimately connected to several types of human cancers, most notably thyroid cancers, making it an attractive therapeutic target for small-molecule kinase inhibitors. Novel approaches, allowing external control of the activity of RET, would be key additions to the signal transduction toolbox. In this work, photoswitchable RET kinase inhibitors based on azo-functionalized pyrazolopyrimidines were developed, enabling photonic control of RET activity. The most promising compound displays excellent switching properties and stability with good inhibitory effect towards RET in cell-free as well as live-cell assays and a significant difference in inhibitory activity between its two photoisomeric forms. As the first reported photoswitchable small-molecule kinase inhibitor, we consider the herein presented effector to be a significant step forward in the development of tools for kinase signal transduction studies with spatiotemporal control over inhibitor concentration in situ.

  20. Kinase Activity Studied in Living Cells Using an Immunoassay

    Science.gov (United States)

    Bavec, Aljos?a

    2014-01-01

    This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

  1. Protein kinase C gamma mutations in spinocerebellar ataxia 14 increase kinase activity and alter membrane targeting

    NARCIS (Netherlands)

    Verbeek, D. S.; Knight, M. A.; Harmison, G. G.; Fischbeck, K. H.; Howell, B. W.

    2005-01-01

    The protein kinase C gamma (PKCgamma) gene is mutated in spinocerebellar ataxia type 14 (SCA14). In this study, we investigated the effects of two SCA14 missense mutations, G118D and C150F, on PKCgamma function. We found that these mutations increase the intrinsic activity of PKCgamma. Direct visual

  2. Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

    DEFF Research Database (Denmark)

    Knecht, Wolfgang; Mikkelsen, N.E.; Clausen, A.R.;

    2009-01-01

    Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2 angstrom resolution s...... structure of DmdNK in complex with gemcitabine shows that the residues Tyr70 and Arg105 play a crucial role in the firm positioning of gemcitabine by extra interactions made by the fluoride atoms. This explains why gemcitabine is a good substrate for Dm-dNK(....

  3. Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

    Energy Technology Data Exchange (ETDEWEB)

    Knecht, Wolfgang [BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby (Denmark); Mikkelsen, Nils Egil [Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Centre, SE-751 24 Uppsala (Sweden); Clausen, Anders Ranegaard [Cell and Organism Biology, Lund University, Soelvegatan 35, SE-22362 Lund (Sweden); Willer, Mette [ZGene A/S, Agern Alle 7, DK-2970 Horsholm (Denmark); Eklund, Hans [Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Centre, SE-751 24 Uppsala (Sweden); Gojkovic, Zoran [ZGene A/S, Agern Alle 7, DK-2970 Horsholm (Denmark); Piskur, Jure, E-mail: Jure.Piskur@cob.lu.se [BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby (Denmark); Cell and Organism Biology, Lund University, Soelvegatan 35, SE-22362 Lund (Sweden)

    2009-05-01

    Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2 A resolution structure of Dm-dNK in complex with gemcitabine shows that the residues Tyr70 and Arg105 play a crucial role in the firm positioning of gemcitabine by extra interactions made by the fluoride atoms. This explains why gemcitabine is a good substrate for Dm-dNK.

  4. Is kinase activity essential for biological functions of BRI1?

    Institute of Scientific and Technical Information of China (English)

    Weihui Xu; Juan Huang; Baohua Li; Jiayang Li; Yonghong Wang

    2008-01-01

    Brassinosteroids (BRs) are a major group of plant hormones that regulate plant growth and development. BRI1, a protein localized to the plasma membrane, functions as a BR receptor and it has been proposed that its kinase activity has an essential role in BR-regulated plant growth and development. Here we report the isolation and molecular characterization of a new allele of bril, bril-301, which shows moderate morphological phenotypes and a reduced response to BRs under normal growth conditions. Sequence analysis identified a two-base alteration from GG to AT, resulting in a conversion of 989G to 9891 in the BRI1 kinase domain. An in vitro assay of kinase activity showed that bril-301 has no detectable autophosphorylation activity or phosphorylation activity towards the BRI1 substrates TTL and BAK1. Furthermore, our results suggest that bril-301, even with extremely impaired kinase activity, still retains partial function in regulating plant growth and development, which raises the question of whether BRI1 kinase activity is essential for BR-mediated growth and development in higher plants.

  5. Protein kinase C controls activation of the DNA integrity checkpoint

    Science.gov (United States)

    Soriano-Carot, María; Quilis, Inma; Bañó, M. Carmen; Igual, J. Carlos

    2014-01-01

    The protein kinase C (PKC) superfamily plays key regulatory roles in numerous cellular processes. Saccharomyces cerevisiae contains a single PKC, Pkc1, whose main function is cell wall integrity maintenance. In this work, we connect the Pkc1 protein to the maintenance of genome integrity in response to genotoxic stresses. Pkc1 and its kinase activity are necessary for the phosphorylation of checkpoint kinase Rad53, histone H2A and Xrs2 protein after deoxyribonucleic acid (DNA) damage, indicating that Pkc1 is required for activation of checkpoint kinases Mec1 and Tel1. Furthermore, Pkc1 electrophoretic mobility is delayed after inducing DNA damage, which reflects that Pkc1 is post-translationally modified. This modification is a phosphorylation event mediated by Tel1. The expression of different mammalian PKC isoforms at the endogenous level in yeast pkc1 mutant cells revealed that PKCδ is able to activate the DNA integrity checkpoint. Finally, downregulation of PKCδ activity in HeLa cells caused a defective activation of checkpoint kinase Chk2 when DNA damage was induced. Our results indicate that the control of the DNA integrity checkpoint by PKC is a mechanism conserved from yeast to humans. PMID:24792164

  6. An N-terminal region of a Myb-like protein is involved in its intracellular localization and activation of a gibberellin-inducible proteinase gene in germinated rice seeds.

    Science.gov (United States)

    Sutoh, Keita; Washio, Kenji; Imai, Ryozo; Wada, Masamitsu; Nakai, Tomonori; Yamauchi, Daisuke

    2015-01-01

    The expression of the gene for a proteinase (Rep1) is upregulated by gibberellins. The CAACTC regulatory element (CARE) of the Rep1 promoter is involved in the gibberellin response. We isolated a cDNA for a CARE-binding protein containing a Myb domain in its carboxyl-terminal region and designated the gene Carboxyl-terminal Myb1 (CTMyb1). This gene encodes two polypeptides of two distinctive lengths, CTMyb1L and CTMyb1S, which include or exclude 213 N-terminal amino acid residues, respectively. CTMyb1S transactivated the Rep1 promoter in the presence of OsGAMyb, but not CTMyb1L. We observed an interaction between CTMyb1S and the rice prolamin box-binding factor (RPBF). A bimolecular fluorescence complex analysis detected the CTMyb1S and RPBF complex in the nucleus, but not the CTMyb1L and RPBF complex. The results suggest that the arrangement of the transfactors is involved in gibberellin-inducible expression of Rep1. PMID:25559339

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

    Science.gov (United States)

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

    2014-08-15

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

  8. Endothelial PI 3-kinase activity regulates lymphocyte diapedesis.

    Science.gov (United States)

    Nakhaei-Nejad, Maryam; Hussain, Amer M; Zhang, Qiu-Xia; Murray, Allan G

    2007-12-01

    Lymphocyte recruitment to sites of inflammation involves a bidirectional series of cues between the endothelial cell (EC) and the leukocyte that culminate in lymphocyte migration into the tissue. Remodeling of the EC F-actin cytoskeleton has been observed after leukocyte adhesion, but the signals to the EC remain poorly defined. We studied the dependence of peripheral blood lymphocyte transendothelial migration (TEM) through an EC monolayer in vitro on EC phosphatidylinositol 3-kinase (PI 3-kinase) activity. Lymphocytes were perfused over cytokine-activated EC using a parallel-plate laminar flow chamber. Inhibition of EC PI 3-kinase activity using LY-294002 or wortmannin decreased lymphocyte TEM (48 +/- 6 or 34 +/- 7%, respectively, vs. control; mean +/- SE; P structure" after intercellular adhesion molecule-1 ligation, whereas this was inhibited by jasplakinolide treatment. A similar fraction of lymphocytes migrated on control or LY-294002-treated EC and localized to interendothelial junctions. However, lymphocytes failed to extend processes below the level of vascular endothelial (VE)-cadherin on LY-294002-treated EC. Together these observations indicate that EC PI 3-kinase activity and F-actin remodeling are required during lymphocyte diapedesis and identify a PI 3-kinase-dependent step following initial separation of the VE-cadherin barrier.

  9. Tumor necrosis factor-alpha induced expression of matrix metalloproteinase-9 through p21-activated Kinase-1

    Directory of Open Access Journals (Sweden)

    Garner Warren

    2009-03-01

    Full Text Available Abstract Background Expressed in embryonic development, matrix metalloprotein-9 (MMP-9 is absent in most of developed adult tissues, but recurs in inflammation during tissue injury, wound healing, tumor formation and metastasis. Expression of MMP-9 is tightly controlled by extracellular cues including pro-inflammatory cytokines and extracellular matrix (ECM. While the pathologic functions of MMP-9 are evident, the intracellular signaling pathways to control its expression are not fully understood. In this study we investigated mechanism of cytokine induced MMP-9 with particular emphasis on the role of p21-activated-kinase-1 (PAK1 and the down stream signaling. Results In response to TNF-alpha or IL-1alpha, PAK1 was promptly activated, as characterized by a sequential phosphorylation, initiated at threonine-212 followed by at threonine-423 in the activation loop of the kinase, in human skin keratinocytes, dermal fibroblasts, and rat hepatic stellate cells. Ectopic expression of PAK1 variants, but not p38 MAP kinase, impaired the TNF-alpha-induced MMP-9 expression, while other MMPs such as MMP-2, -3 and -14 were not affected. Activation of Jun N-terminal kinase (JNK and NF-kappaB has been demonstrated to be essential for MMP-9 expression. Expression of inactive PAK1 variants impaired JNK but not NF-kappaB activation, which consequently suppressed the 5'-promoter activities of the MMP-9 gene. After the cytokine-induced phosphorylation, both ectopically expressed and endogenous PAK1 proteins were promptly accumulated even in the condition of suppressing protein synthesis, suggesting the PAK1 protein is stabilized upon TNF-alpha stimulation. Stabilization of PAK1 protein by TNF-alpha treatment is independent of the kinase catalytic activity and p21 GTPase binding capacities. In contrast to epithelial cells, mesenchymal cells require 3-dimensional type-I collagen in response to TNF-alpha to massively express MMP-9. The collagen effect is mediated, in

  10. Enzymatic assay for calmodulins based on plant NAD kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.

    1984-01-01

    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  11. A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B.

    Science.gov (United States)

    Nijenhuis, Wilco; von Castelmur, Eleonore; Littler, Dene; De Marco, Valeria; Tromer, Eelco; Vleugel, Mathijs; van Osch, Maria H J; Snel, Berend; Perrakis, Anastassis; Kops, Geert J P L

    2013-04-15

    The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.

  12. The effects of interleukin-1b in modulating osteoclast-conditioned medium’s influence on gelatinases in chondrocytes through mitogen-activated protein kinases

    Institute of Scientific and Technical Information of China (English)

    Xiao-Xiao Cai

    2015-01-01

    Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase (MAPK) signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1b (IL-1b)-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1b induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1b restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-1b restores gelatinase activity through MAPK inhibitors;this information can help to increase the understanding of the gelatinase modulation in articular cartilage.

  13. In silico analysis reveals 75 members of mitogen-activated protein kinase kinase kinase gene family in rice.

    Science.gov (United States)

    Rao, Kudupudi Prabhakara; Richa, Tambi; Kumar, Kundan; Raghuram, Badmi; Sinha, Alok Krishna

    2010-06-01

    Mitogen-Activated Protein Kinase Kinase Kinases (MAPKKKs) are important components of MAPK cascades, which are universal signal transduction modules and play important role in plant growth and development. In the sequenced Arabidopsis genome 80 MAPKKKs were identified and currently being analysed for its role in different stress. In rice, economically important monocot cereal crop only five MAPKKKs were identified so far. In this study using computational analysis of sequenced rice genome we have identified 75 MAPKKKs. EST hits and full-length cDNA sequences (from KOME or Genbank database) of 75 MAPKKKs supported their existence. Phylogenetic analyses of MAPKKKs from rice and Arabidopsis have classified them into three subgroups, which include Raf, ZIK and MEKK. Conserved motifs in the deduced amino acid sequences of rice MAPKKKs strongly supported their identity as members of Raf, ZIK and MEKK subfamilies. Further expression analysis of the MAPKKKs in MPSS database revealed that their transcripts were differentially regulated in various stress and tissue-specific libraries.

  14. Overinhibition of Mitogen-Activated Protein Kinase Inducing Tau Hyperphosphorylation

    Institute of Scientific and Technical Information of China (English)

    LI Hong-lian; CHEN Juan; LIU Shi-jie; ZHANG Jia-yu; WANG Qun; WANG Jian-zhi

    2005-01-01

    To reveal the relationship between mitogen-activated protein kinase (MAPK) and tau phosphorylation, we used different concentration of PD98059, an inhibitor of MEK (MAPK kinase), to treat mice neuroblastma (N2a) cell line for 6 h. It showed that the activity of MAPK decreased in a dose-dependent manner. But Western blot and immunofluorescence revealed that just when the cells were treated with 16 μmol/L PD98059, tau was hyperphosphorylated at Ser396/404 and Ser199/202 sites. We obtained the conclusion that overinhibited MAPK induced tau hyperphosphorylation at Ser396/404 and Ser199/202 sites.

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

    Directory of Open Access Journals (Sweden)

    Bernardo Ramírez-Zavala

    Full Text Available Depending on the environmental conditions, the pathogenic yeast Candida albicans can undergo different developmental programs, which are controlled by dedicated transcription factors and upstream signaling pathways. C. albicans strains that are homozygous at the mating type locus can switch from the normal yeast form (white to an elongated cell type (opaque, which is the mating-competent form of this fungus. Both white and opaque cells use the Ste11-Hst7-Cek1/Cek2 MAP kinase signaling pathway to react to the presence of mating pheromone. However, while opaque cells employ the transcription factor Cph1 to induce the mating response, white cells recruit a different downstream transcription factor, Tec1, to promote the formation of a biofilm that facilitates mating of opaque cells in the population. The switch from the white to the opaque cell form is itself induced by environmental signals that result in the upregulation of the transcription factor Wor1, the master regulator of white-opaque switching. To get insight into the upstream signaling pathways controlling the switch, we expressed all C. albicans protein kinases from a tetracycline-inducible promoter in a switching-competent strain. Screening of this library of strains showed that a hyperactive form of Ste11 lacking its N-terminal domain (Ste11(ΔN467 efficiently stimulated white cells to switch to the opaque phase, a behavior that did not occur in response to pheromone. Ste11(ΔN467-induced switching specifically required the downstream MAP kinase Cek1 and its target transcription factor Cph1, but not Cek2 and Tec1, and forced expression of Cph1 also promoted white-opaque switching in a Wor1-dependent manner. Therefore, depending on the activation mechanism, components of the pheromone-responsive MAP kinase pathway can be reconnected to stimulate an alternative developmental program, switching of white cells to the mating-competent opaque phase.

  16. The carboxy-terminal tail of pyruvate dehydrogenase kinase 2 is required for the kinase activity.

    Science.gov (United States)

    Klyuyeva, Alla; Tuganova, Alina; Popov, Kirill M

    2005-10-18

    Pyruvate dehydrogenase kinase 2 (PDK2) is a prototypical mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase complex. Recent structural studies have established that PDK2 consists of a catalytic core built of the B and K domains and the relatively long amino and carboxyl tails of unknown function. Here, we show that the carboxy-terminal truncation variants of PDK2 display a greatly diminished capacity for phosphorylation of holo-PDC. This effect is due largely to the inability of the transacetylase component of PDC to promote the phosphorylation reaction catalyzed by the truncated PDK2 variants. Furthermore, the truncated forms of PDK2 bind poorly to the lipoyl-bearing domain(s) provided by the transacetylase component. Taken together, these data strongly suggest that the carboxyl tails of PDK isozymes contribute to the lipoyl-bearing domain-binding site of the kinase molecule. We also show that the carboxyl tails derived from isozymes PDK1, PDK3, and PDK4 are capable of supporting the kinase activity of the kinase core derived from PDK2 as well as binding of the respective PDK2 chimeras to the lipoyl-bearing domain. Furthermore, the chimera carrying the carboxyl tail of PDK3 displays a stronger response to the addition of the transacetylase component along with a better binding to the lipoyl-bearing domain, suggesting that, at least in part, the differences in the amino acid sequences of the carboxyl tails account for the differences between PDK isozymes. PMID:16216081

  17. All-trans retinoic acid modulates mitogen-activated protein kinase pathway activation in human scleral fibroblasts through retinoic acid receptor beta

    Science.gov (United States)

    Huo, Lijun; Cui, Dongmei; Yang, Xiao; Gao, Zhenya; Trier, Klaus

    2013-01-01

    Purpose All-trans retinoic acid (ATRA) is known to inhibit the proliferation of human scleral fibroblasts (HSFs) and to modulate the scleral intercellular matrix composition, and may therefore serve as a mediator for controlling eye growth. Cell proliferation is regulated by the mitogen-activated protein kinase (MAPK) pathway. The aim of the current study was to investigate whether changed activation of the MAPK pathway could be involved in the response of HSFs exposed to ATRA. Methods HSFs were cultured in Dulbecco Modified Eagle's Medium/F12 (DMEM/F12) and exposed to 1 μmol/l ATRA for 10 min, 30 min, 1 h, 8 h, or 24 h. The activation of extracellular signal-regulated kinase (ERK 1/2), p38, and c-Jun N-terminal kinase (JNK) in HSFs was assessed with western blot analysis and immunocytofluorescence. Results After exposure to ATRA for 24 h, the HSFs appeared shrunken and thinner than the control cells. The intercellular spaces were wider, and the HSFs appeared less numerous than in the control culture. Western blot showed decreased activation of ERK 1/2 in the HSFs from 30 min (p=0.01) to 24 h (p<0.01) after the start of exposure to ATRA, and increased activation of the JNK protein from 10 to 30 min (p<0.01) after the start of exposure to ATRA. Indirect immunofluorescence confirmed changes in activation of ERK 1/2 and JNK in HSFs exposed to ATRA. No change in activation of p38 in HSFs was observed after exposure to ATRA. Pretreatment of the HSFs with LE135, an antagonist of retinoic acid receptor beta (RARβ), abolished the ATRA-induced changes inactivation of ERK 1/2 and JNK. Conclusions ATRA inhibits HSF proliferation by a mechanism associated with modulation of ERK 1/2 and JNK activation and depends on stimulation of retinoic acid receptor beta. PMID:23946634

  18. Protein kinase D activity controls endothelial nitric oxide synthesis.

    Science.gov (United States)

    Aicart-Ramos, Clara; Sánchez-Ruiloba, Lucía; Gómez-Parrizas, Mónica; Zaragoza, Carlos; Iglesias, Teresa; Rodríguez-Crespo, Ignacio

    2014-08-01

    Vascular endothelial growth factor (VEGF) regulates key functions of the endothelium, such as angiogenesis or vessel repair in processes involving endothelial nitric oxide synthase (eNOS) activation. One of the effector kinases that become activated in endothelial cells upon VEGF treatment is protein kinase D (PKD). Here, we show that PKD phosphorylates eNOS, leading to its activation and a concomitant increase in NO synthesis. Using mass spectrometry, we show that the purified active kinase specifically phosphorylates recombinant eNOS on Ser1179. Treatment of endothelial cells with VEGF or phorbol 12,13-dibutyrate (PDBu) activates PKD and increases eNOS Ser1179 phosphorylation. In addition, pharmacological inhibition of PKD and gene silencing of both PKD1 and PKD2 abrogate VEGF signaling, resulting in a clear diminished migration of endothelial cells in a wound healing assay. Finally, inhibition of PKD in mice results in an almost complete disappearance of the VEGF-induced vasodilatation, as monitored through determination of the diameter of the carotid artery. Hence, our data indicate that PKD is a new regulatory kinase of eNOS in endothelial cells whose activity orchestrates mammalian vascular tone. PMID:24928905

  19. Mitogen-activated protein kinase signaling in plants

    DEFF Research Database (Denmark)

    Rodriguez, Maria Cristina Suarez; Petersen, Morten; Mundy, John

    2010-01-01

    of substrate proteins, whose altered activities mediate a wide array of responses, including changes in gene expression. Cascades may share kinase components, but their signaling specificity is maintained by spaciotemporal constraints and dynamic protein-protein interactions and by mechanisms that include......Eukaryotic mitogen-activated protein kinase (MAPK) cascades have evolved to transduce environmental and developmental signals into adaptive and programmed responses. MAPK cascades relay and amplify signals via three types of reversibly phosphorylated kinases leading to the phosphorylation...... crossinhibition, feedback control, and scaffolding. Plant MAPK cascades regulate numerous processes, including stress and hormonal responses, innate immunity, and developmental programs. Genetic analyses have uncovered several predominant MAPK components shared by several of these processes including...

  20. Toll 样受体2介导的 JNK 信号分子在小鼠支气管哮喘发病中的作用机制%Mechanism of c-Jun N-terminal kinase mediated by Toll like receptor 2 in murine asthma

    Institute of Scientific and Technical Information of China (English)

    沈佩婷; 方磊; 吴惠梅; 沈启英; 何芳; 刘荣玉

    2015-01-01

    目的:探讨 Toll 样受体2(TLR2)介导的 c-Jun 氨基末端激酶(JNK)信号分子参与小鼠支气管哮喘发病的作用机制。方法健康 SPF 级 C57(TLR2野生型)鼠和 TLR2基因缺失(TLR2-/-)鼠各14只,按随机数字表法分为4组:C57对照组、C57哮喘组、TLR2-/-对照组、TLR2-/-哮喘组,每组7只,哮喘组以卵清蛋白(OVA)腹腔注射联合雾化吸入致敏和激发建立哮喘模型,对照组以生理盐水代替 OVA致敏和激发。利用免疫组织化学染色技术( ABC 法)检测TLR2蛋白在 C57对照组、C57哮喘组肺内的表达差异,JNK及磷酸化 JNK(P-JNK)蛋白表达在各组肺内的表达差异。结果 HE 染色提示较其余3组,C57哮喘组有较明显的炎症细胞浸润及呼吸道平滑肌增生。以平均吸光度(mA)衡量各组织蛋白相对表达量,免疫组化结果提示 TLR2蛋白在C57哮喘组表达显著高于 C57对照组(P <0.01),JNK 蛋白在各组的表达差异无统计学意义,P-JNK 蛋白在 C57哮喘组肺组织的表达量显著高于 C57对照组、TLR2-/-哮喘组、TLR2-/-对照组(F =43.261,P <0.01)。结论 TLR2介导的 JNK 信号分子通路可能参与了支气管哮喘的发病过程。%Objective To explore the mechanism of c-Jun N-terminal kinase mediated by Toll like receptor 2 in murine asthma. Methods 14 healthy SPF grade C57 wild-type mice and 14 TLR2 knockout (TLR2 - / - ) mice were randomly divided into four groups: C57 control group, C57 asthma group, TLR2 - / - control group, TLR2 - / - asth-ma group (n = 7). We utilized intraperitoneal injection combined with inhalation of ovalbumin (OVA) to sensitize and challenge the mice, thus establishing the experimental models of asthma. Meanwhile, the control group received normal saline instead of OVA. The protein expression of TLR2 was detected by immunohistochemistry(ABC meth-od) in C57 control group and C57 asthma group,as well as JNK and phosphorylation c-Jun(P-JNK) between each group. Results In C57

  1. Sequence-specific and general transcriptional activation by the bovine papillomavirus-1 E2 trans-activator require an N-terminal amphipathic helix-containing E2 domain.

    OpenAIRE

    Haugen, T H; Turek, L P; Mercurio, F M; Cripe, T P; Olson, B J; Anderson, R D; D. Seidl; Karin, M; Schiller, J.

    1988-01-01

    The sequence-specific trans-activator protein of bovine papillomavirus (BPV)-1, E2, strongly increases transcription at promoters containing papillomaviral ACCG(N)4CGGT (E2P) cis motifs, but can also activate a wide range of co-transfected promoters without E2P cores to a lower extent. Analysis of multiple E2 mutants in transfected cells revealed that the C-terminal DNA binding E2 domain binds to the E2P cis sequences in the form of pre-existing nuclear dimers. The DNA binding function of E2 ...

  2. Arabidopsis MKS1 is involved in basal immunity and requires an intact N-terminal domain for proper function.

    Directory of Open Access Journals (Sweden)

    Klaus Petersen

    Full Text Available BACKGROUND: Innate immune signaling pathways in animals and plants are regulated by mitogen-activated protein kinase (MAPK cascades. MAP kinase 4 (MPK4 functions downstream of innate immune receptors via a nuclear substrate MKS1 to regulate the activity of the WRKY33 transcription factor, which in turn controls the production of anti-microbial phytoalexins. METHODOLOGY/PRINCIPAL FINDINGS: We investigate the role of MKS1 in basal resistance and the importance of its N- and C-terminal domains for MKS1 function. We used the information that mks1 loss-of-function partially suppresses the mpk4 loss-of-function phenotype, and that transgenic expression of functional MKS1 in mpk4/mks1 double mutants reverted the mpk4 dwarf phenotype. Transformation of mks1/mpk4 with mutant versions of MKS1 constructs showed that a single amino acid substitution in a putative MAP kinase docking domain, MKS1-L32A, or a truncated MKS1 version unable to interact with WRKY33, were deficient in reverting the double mutant to the mpk4 phenotype. These results demonstrate functional requirement in MKS1 for the interaction with MPK4 and WRKY33. In addition, nuclear localization of MKS1 was shown to depend on an intact N-terminal domain. Furthermore, loss-of-function mks1 mutants exhibited increased susceptibility to strains of Pseudomonas syringae and Hyaloperonospora arabidopsidis, indicating that MKS1 plays a role in basal defense responses. CONCLUSIONS: Taken together, our results indicate that MKS1 function and subcellular location requires an intact N-terminus important for both MPK4 and WRKY33 interactions.

  3. Structural modeling of the N-terminal signal–receiving domain of IκBα

    Directory of Open Access Journals (Sweden)

    Samira eYazdi

    2015-06-01

    Full Text Available The transcription factor nuclear factor-κB (NF-κB exerts essential roles in many biological processes including cell growth, apoptosis and innate and adaptive immunity. The NF-kB inhibitor (IκBα retains NF-κB in the cytoplasm and thus inhibits nuclear localization of NF-κB and its association with DNA. Recent protein crystal structures of the C-terminal part of IκBα in complex with NF-κB provided insights into the protein-protein interactions but could not reveal structural details about the N-terminal signal receiving domain (SRD. The SRD of IκBα contains a degron, formed following phosphorylation by IκB kinases (IKK. In current protein X-ray structures, however, the SRD is not resolved and assumed to be disordered. Here, we combined secondary structure annotation and domain threading followed by long molecular dynamics (MD simulations and showed that the SRD possesses well-defined secondary structure elements. We show that the SRD contains 3 additional stable α-helices supplementing the six ARDs present in crystallized IκBα. The IκBα/NF-κB protein-protein complex remained intact and stable during the entire simulations. Also in solution, free IκBα retains its structural integrity. Differences in structural topology and dynamics were observed by comparing the structures of NF-κB free and NF-κB bound IκBα-complex. This study paves the way for investigating the signaling properties of the SRD in the IκBα degron. A detailed atomic scale understanding of molecular mechanism of NF-κB activation, regulation and the protein-protein interactions may assist to design and develop novel chronic inflammation modulators.

  4. Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

    Energy Technology Data Exchange (ETDEWEB)

    Couto, Sheila G. [Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador Sao-carlense 400, C.P. 369, 13560-970, Sao Carlos, SP (Brazil); Grupo de Biofisica e Fisica Aplicada a Medicina, Instituto de Fisica, Universidade Federal de Goias, Campus Samambaia, C.P. 131, 74001-970, Goiania, GO (Brazil); Cristina Nonato, M. [Laboratorio de Cristalografia de Proteinas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Av. do Cafe S/N, 14040-903, Ribeirao Preto, SP (Brazil); Costa-Filho, Antonio J., E-mail: ajcosta@ffclrp.usp.br [Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador Sao-carlense 400, C.P. 369, 13560-970, Sao Carlos, SP (Brazil); Departamento de Fisica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Av. Bandeirantes 3900, 14040-901, Ribeirao Preto, SP (Brazil)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer EcDHODH is a membrane-associated enzyme and a promising target for drug design. Black-Right-Pointing-Pointer Enzyme's N-terminal extension is responsible for membrane association. Black-Right-Pointing-Pointer N-terminal works as a molecular lid regulating access to the protein interior. -- Abstract: Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme's N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

  5. Structural and functional insights into the role of the N-terminal Mps1 TPR domain in the SAC (spindle assembly checkpoint).

    Science.gov (United States)

    Thebault, Philippe; Chirgadze, Dimitri Y; Dou, Zhen; Blundell, Tom L; Elowe, Sabine; Bolanos-Garcia, Victor M

    2012-12-15

    The SAC (spindle assembly checkpoint) is a surveillance system that ensures the timely and accurate transmission of the genetic material to offspring. The process implies kinetochore targeting of the mitotic kinases Bub1 (budding uninhibited by benzamidine 1), BubR1 (Bub1 related) and Mps1 (monopolar spindle 1), which is mediated by the N-terminus of each kinase. In the present study we report the 1.8 Å (1 Å=0.1 nm) crystal structure of the TPR (tetratricopeptide repeat) domain in the N-terminal region of human Mps1. The structure reveals an overall high similarity to the TPR motif of the mitotic checkpoint kinases Bub1 and BubR1, and a number of unique features that include the absence of the binding site for the kinetochore structural component KNL1 (kinetochore-null 1; blinkin), and determinants of dimerization. Moreover, we show that a stretch of amino acids at the very N-terminus of Mps1 is required for dimer formation, and that interfering with dimerization results in mislocalization and misregulation of kinase activity. The results of the present study provide an important insight into the molecular details of the mitotic functions of Mps1 including features that dictate substrate selectivity and kinetochore docking.

  6. Redox Regulation of the AMP-Activated Protein Kinase

    OpenAIRE

    Yingying Han; Qilong Wang; Ping Song; Yi Zhu; Ming-Hui Zou

    2010-01-01

    Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death. Objectives The aim of this study is to determine if AMP-activated protein kinase (AMPK), a cellular energy sensor, is activated by oxidants generated by Berberine in endothelial cells (EC). Methods Bovine aortic endothelial cells (BAEC) were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation. Results In BAEC, Berberine caused a dos...

  7. Mitogen activated protein kinases: a role in inflammatory bowel disease?

    DEFF Research Database (Denmark)

    Broom, O J; Widjaya, B; Troelsen, J;

    2009-01-01

    Since their discovery more than 15 years ago, the mitogen activated protein kinases (MAPK) have been implicated in an ever-increasingly diverse array of pathways, including inflammatory signalling cascades. Inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn's disease...... and their related signalling proteins in influencing the progression of IBD....

  8. Janus Kinase 2: An Epigenetic 'Writer' that Activates Leukemogenic Genes

    Institute of Scientific and Technical Information of China (English)

    Jin He; Yi Zhang

    2010-01-01

    @@ Activation of Janus kinase 2 (JAK2) plays a critical role in normal hematopoiesis and leukemogenesis. Dawson et al. (2009; JAK2 phosphorylates histone H3Y41 and excludes Hplalpha from chromatin. Nature 461, 819-822) report that JAK2 performs this function by displacing the heterochromatin protein HP1α from chromatin through phosphorylation of histone H3.

  9. The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Pozo-Yauner, Luis del, E-mail: ldelpozo@inmegen.gob.mx [Instituto Nacional de Medicina Genómica, Periférico Sur No. 4809, Col. Arenal Tepepan, Delegación Tlalpan, México, D.F. C.P. 14610 (Mexico); Wall, Jonathan S. [Departments of Radiology and Medicine, The University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville, TN (United States); González Andrade, Martín [Instituto Nacional de Medicina Genómica, Periférico Sur No. 4809, Col. Arenal Tepepan, Delegación Tlalpan, México, D.F. C.P. 14610 (Mexico); Sánchez-López, Rosana [Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa Cuernavaca, Morelos C.P. 62210 (Mexico); Rodríguez-Ambriz, Sandra L. [Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional, Calle CEPROBI No. 8, Col. San Isidro, Yautepec, Morelos C.P. 62731 (Mexico); Pérez Carreón, Julio I. [Instituto Nacional de Medicina Genómica, Periférico Sur No. 4809, Col. Arenal Tepepan, Delegación Tlalpan, México, D.F. C.P. 14610 (Mexico); and others

    2014-01-10

    Highlights: •We evaluated the impact of mutations in the N-terminal strand of 6aJL2 protein. •Mutations destabilized the protein in a position-dependent manner. •Destabilizing mutations accelerated the fibrillogenesis by shortening the lag time. •The effect on the kinetic of fibril elongation by seeding was of different nature. •The N-terminal strand is buried in the fibrillar state of 6aJL2 protein. -- Abstract: It has been suggested that the N-terminal strand of the light chain variable domain (V{sub L}) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V{sub L} protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.

  10. Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation.

    OpenAIRE

    Bunone, G; Briand, P A; Miksicek, R J; Picard, D.

    1996-01-01

    The estrogen receptor (ER) can be activated as a transcription factor either by binding of cognate estrogenic ligand or, indirectly, by a variety of other extracellular signals. As a first step towards elucidating the mechanism of 'steroid-independent activation' of the ER by the epidermal growth factor (EGF), we have mapped the ER target domain and determined the signaling pathway. We show that the N-terminal transcriptional activation function AF-1, but not the C-terminal AF-2, is necessary...

  11. Activated type I TGFbeta receptor (Alk5) kinase confers enhancedsurvival to mammary epithelial cells and accelerates mammary tumorprogression

    Energy Technology Data Exchange (ETDEWEB)

    Muraoka-Cook, Rebecca S.; Shin, Incheol; Yi, Jae Youn; Easterly,Evangeline; Barcellos-Hoff, Mary Helen; Yingling, Jonathan M.; Zent, Roy; Arteaga, Carlos L.

    2005-01-02

    The transforming growth factor-betas (TGF{beta}s) are members of a large superfamily of pleiotropic cytokines that also includes the activins and the bone morphogenetic proteins (BMPs). Members of the TGF{beta} family regulate complex physiological processes such cell proliferation, differentiation, adhesion, cell-cell and cell-matrix interactions, motility, and cell death, among others (Massague, 1998). Dysregulation of TGF{beta} signaling contributes to several pathological processes including cancer, fibrosis, and auto-immune disorders (Massague et al., 2000). The TGF{beta}s elicit their biological effects by binding to type II and type I transmembrane receptor serine-threonine kinases (T{beta}RII and T{beta}RI) which, in turn, phosphorylated Smad 2 and Smad 3. Phosphorylated Smad 2/3 associate with Smad 4 and, as a heteromeric complex, translocate to the nucleus where they regulate gene transcription. The inhibitory Smad7 down regulates TGF{beta} signaling by binding to activated T{beta}RI and interfering with its ability to phosphorylate Smad 2/3 (Derynck and Zhang, 2003; Shi and Massague, 2003). Signaling is also regulated by Smad proteolysis. TGF{beta} receptor-mediated activation results in multi-ubiquitination of Smad 2 in the nucleus and subsequent degradation of Smad 2 by the proteasome (Lo and Massague, 1999). Activation of TGF{beta} receptors also induces mobilization of a Smad 7-Smurf complex from the nucleus to the cytoplasm; this complex recognizes the activated receptors and mediates their ubiquitination and internalization via caveolin-rich vesicles, leading to termination of TGF{beta} signaling (Di Guglielmo et al., 2003). Other signal transducers/pathways have been implicated in TGF{beta} actions. These include the extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (Jnk), p38 mitogen-activated protein kinase (MAPK), protein phosphatase PP2A, phosphatidylinositol-3 kinase (PI3K), and the family of Rho GTPases [reviewed in

  12. Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition

    Science.gov (United States)

    Chang, Ching Ching; Few, Ling Ling; Konrad, Manfred; See Too, Wei Cun

    2016-01-01

    Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis. PMID:27149373

  13. Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation

    Institute of Scientific and Technical Information of China (English)

    Hai Jiang; Jianchun Wu; Chen He; Wending Yang; Honglin Li

    2009-01-01

    Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-kB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdkl activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chkl and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.

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

    DEFF Research Database (Denmark)

    Wang, Zhipeng; Fu, Meng; Wang, Lifeng;

    2013-01-01

    PAK1 plays an important role in proliferation and tumorigenesis, at least partially by promoting Erk phosphorylation of c-Raf (Ser 338) or Mek1 (Ser 298). We observed now that overexpression of a kinase-dead mutant form of PAK1 increased phosphorylation of Mek1/2 (Ser 217/221) and Erk (Thr 202/Tyr...

  15. Ethanol Metabolism Activates Cell Cycle Checkpoint Kinase, Chk2

    Science.gov (United States)

    Clemens, Dahn L.; Mahan Schneider, Katrina J.; Nuss, Robert F.

    2011-01-01

    Chronic ethanol abuse results in hepatocyte injury and impairs hepatocyte replication. We have previously shown that ethanol metabolism results in cell cycle arrest at the G2/M transition, which is partially mediated by inhibitory phosphorylation of the cyclin-dependent kinase, Cdc2. To further delineate the mechanisms by which ethanol metabolism mediates this G2/M arrest, we investigated the involvement of upstream regulators of Cdc2 activity. Cdc2 is activated by the phosphatase Cdc25C. The activity of Cdc25C can, in turn, be regulated by the checkpoint kinase, Chk2, which is regulated by the kinase ataxia telangiectasia mutated (ATM). To investigate the involvement of these regulators of Cdc2 activity, VA-13 cells, which are Hep G2 cells modified to efficiently express alcohol dehydrogenase, were cultured in the presence or absence of 25 mM ethanol. Immunoblots were performed to determine the effects of ethanol metabolism on the activation of Cdc25C, Chk2, and ATM. Ethanol metabolism increased the active forms of ATM, and Chk2, as well as the phosphorylated form of Cdc25C. Additionally, inhibition of ATM resulted in approximately 50% of the cells being rescued from the G2/M cell cycle arrest, and ameliorated the inhibitory phosphorylation of Cdc2. Our findings demonstrate that ethanol metabolism activates ATM. ATM can activate the checkpoint kinase Chk2, resulting in phosphorylation of Cdc25C, and ultimately in the accumulation of inactive Cdc2. This may, in part, explain the ethanol metabolism-mediated impairment in hepatocyte replication, which may be important in the initiation and progression of alcoholic liver injury. PMID:21924579

  16. Timeless links replication termination to mitotic kinase activation.

    Science.gov (United States)

    Dheekollu, Jayaraju; Wiedmer, Andreas; Hayden, James; Speicher, David; Gotter, Anthony L; Yen, Tim; Lieberman, Paul M

    2011-05-06

    The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim) associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1). Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication.

  17. Timeless links replication termination to mitotic kinase activation.

    Directory of Open Access Journals (Sweden)

    Jayaraju Dheekollu

    Full Text Available The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1. Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication.

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

    Science.gov (United States)

    Temmerman, Koen; Simon, Bertrand; Wilmanns, Matthias

    2013-11-01

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

  19. Regulation of Akt/PKB activity by P21-activated Kinase in Cardiomyocytes

    OpenAIRE

    Mao, Kai; Kobayashi, Satoru; Jaffer, Zahara M.; Huang, Yuan; Volden, Paul; Chernoff, Jonathan; Liang, Qiangrong

    2007-01-01

    Akt/PKB is a critical regulator of cardiac function and morphology, and its activity is governed by dual phosphorylation at active loop (Thr308) by phosphoinositide-dependent protein kinase-1 (PDK1) and at carboxyl-terminal hydrophobic motif (Ser473) by a putative PDK2. P21-activated kinase-1 (Pak1) is a serine/threonine protein kinase implicated in the regulation of cardiac hypertrophy and contractility, and was shown previously to activate Akt through an undefined mechanism. Here we report ...

  20. Ca2+/calmodulin-dependent protein kinase kinase is not involved in hypothalamic AMP-activated protein kinase activation by neuroglucopenia.

    Directory of Open Access Journals (Sweden)

    Junji Kawashima

    Full Text Available Hypoglycemia and neuroglucopenia stimulate AMP-activated protein kinase (AMPK activity in the hypothalamus and this plays an important role in the counterregulatory responses, i.e. increased food intake and secretion of glucagon, corticosterone and catecholamines. Several upstream kinases that activate AMPK have been identified including Ca(2+/Calmodulin-dependent protein kinase kinase (CaMKK, which is highly expressed in neurons. However, the involvement of CaMKK in neuroglucopenia-induced activation of AMPK in the hypothalamus has not been tested. To determine whether neuroglucopenia-induced AMPK activation is mediated by CaMKK, we tested whether STO-609 (STO, a CaMKK inhibitor, would block the effects of 2-deoxy-D-glucose (2DG-induced neuroglucopenia both ex vivo on brain sections and in vivo. Preincubation of rat brain sections with STO blocked KCl-induced α1 and α2-AMPK activation but did not affect AMPK activation by 2DG in the medio-basal hypothalamus. To confirm these findings in vivo, STO was pre-administrated intracerebroventricularly (ICV in rats 30 min before 2DG ICV injection (40 µmol to induce neuroglucopenia. 2DG-induced neuroglucopenia lead to a significant increase in glycemia and food intake compared to saline-injected control rats. ICV pre-administration of STO (5, 20 or 50 nmol did not affect 2DG-induced hyperglycemia and food intake. Importantly, activation of hypothalamic α1 and α2-AMPK by 2DG was not affected by ICV pre-administration of STO. In conclusion, activation of hypothalamic AMPK by 2DG-induced neuroglucopenia is not mediated by CaMKK.

  1. Creatine kinase activity in dogs with experimentally induced acute inflammation

    Directory of Open Access Journals (Sweden)

    Dimitrinka Zapryanova

    2013-01-01

    Full Text Available The main purpose of this study was to investigate the effect of acute inflammation on total creatine kinase (CK activity in dogs. In these animals, CK is an enzyme found predominantly in skeletal muscle and significantly elevated serum activity is largely associated with muscle damage. Plasma increases in dogs are associated with cell membrane leakage and will therefore be seen in any condition associated with muscular inflammation. The study was induced in 15 mongrel male dogs (n=9 in experimental group and n=6 in control group at the age of two years and body weight 12-15 kg. The inflammation was reproduced by inoculation of 2 ml turpentine oil subcutaneously in lumbar region. The plasma activity of creatine kinase was evaluated at 0, 6, 24, 48, 72 hours after inoculation and on days 7, 14 and 21 by a kit from Hospitex Diagnostics. In the experimental group, the plasma concentrations of the CK-activity were increased at the 48th hour (97.48±6.92 U/L and remained significantly higher (p<0.05 at the 72 hour (97.43±2.93 U/L compared to the control group (77.08±5.27 U/L. The results of this study suggest that the evaluation of creatine kinase in dogs with experimentally induced acute inflammation has a limited diagnostic value. It was observed that the creatine kinase activity is slightly affected by the experimentally induced acute inflammation in dogs.

  2. Leishmania amazonensis: PKC-like protein kinase modulates the (Na++K+)ATPase activity.

    Science.gov (United States)

    Almeida-Amaral, Elmo Eduardo de; Caruso-Neves, Celso; Lara, Lucienne Silva; Pinheiro, Carla Mônica; Meyer-Fernandes, José Roberto

    2007-08-01

    The present study aimed to identify the presence of protein kinase C-like (PKC-like) in Leishmania amazonensis and to elucidate its possible role in the modulation of the (Na(+)+K(+))ATPase activity. Immunoblotting experiments using antibody against a consensus sequence (Ac 543-549) of rabbit protein kinase C (PKC) revealed the presence of a protein kinase of 80 kDa in L. amazonensis. Measurements of protein kinase activity showed the presence of both (Ca(2+)-dependent) and (Ca(2+)-independent) protein kinase activity in plasma membrane and cytosol. Phorbol ester (PMA) activation of the Ca(2+)-dependent protein kinase stimulated the (Na(+)+K(+))ATPase activity, while activation of the Ca(2+)-independent protein kinase was inhibitory. Both effects of protein kinase on the (Na(+)+K(+))ATPase of the plasma membrane were lower than that observed in intact cells. PMA induced the translocation of protein kinase from cytosol to plasma membrane, indicating that the maximal effect of protein kinase on the (Na(+)+K(+))ATPase activity depends on the synergistic action of protein kinases from both plasma membrane and cytosol. This is the first demonstration of a protein kinase activated by PMA in L. amazonensis and the first evidence for a possible role in the regulation of the (Na(+)+K(+))ATPase activity in this trypanosomatid. Modulation of the (Na(+)+K(+))ATPase by protein kinase in a trypanosomatid opens up new possibilities to understand the regulation of ion homeostasis in this parasite. PMID:17475255

  3. Association of Common Genetic Variants in Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 with Type 2 Diabetes Mellitus in a Chinese Han Population

    Institute of Scientific and Technical Information of China (English)

    Ting-Ting Li; Hong Qiao; Hui-Xin Tong; Tian-Wei Zhuang; Tong-Tong Wang

    2016-01-01

    Background:A study has identified several novel susceptibility variants of the mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) gene for type 2 diabetes mellitus (T2DM) within the German population.Among the variants,five single nucleotide polymorphisms (SNPs) of MAP4K4 (rs1003376,rs11674694,rs2236935,rs2236936,and rs6543087) showed significant association with T2DM or diabetes-related quantitative traits.We aimed to evaluate whether common SNPs in the MAP4K4 gene were associated with T2DM in the Chinese population.Methods:Five candidate SNPs were genotyped in 996 patients newly diagnosed with T2DM and in 976 control subjects,using the SNPscanTM method.All subjects were recruited from the Second Affiliated Hospital,Harbin Medical University from October 2010 to September 2013.We evaluated the T2DM risk conferred by individual SNPs and haplotypes using logistic analysis,and the association between the five SNPs and metabolic traits in the subgroups.Results:Of the five variants,SNP rs2236935T/C was significantly associated with T2DM in this study population (odds ratio =1.293;95% confidence interval:1.034-1.619,P =0.025).In addition,among the controls,rs 1003376 was significantly associated with an increased body mass index (P =0.045) and homeostatic model assessment-insulin resistance (P =0.037).Conclusions:MAP4K4 gene is associated with T2DM in a Chinese Han population,and MAP4K4 gene variants may contribute to the risk toward the development of T2DM.

  4. Genome-wide identification and analysis of expression profiles of maize mitogen-activated protein kinase kinase kinase.

    Science.gov (United States)

    Kong, Xiangpei; Lv, Wei; Zhang, Dan; Jiang, Shanshan; Zhang, Shizhong; Li, Dequan

    2013-01-01

    Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction model in animals, yeast and plants. Plant MAPK cascades have been implicated in development and stress responses. Although MAPKKKs have been investigated in several plant species including Arabidopsis and rice, no systematic analysis has been conducted in maize. In this study, we performed a bioinformatics analysis of the entire maize genome and identified 74 MAPKKK genes. Phylogenetic analyses of MAPKKKs from maize, rice and Arabidopsis have classified them into three subgroups, which included Raf, ZIK and MEKK. Evolutionary relationships within subfamilies were also supported by exon-intron organizations and the conserved protein motifs. Further expression analysis of the MAPKKKs in microarray databases revealed that MAPKKKs were involved in important signaling pathways in maize different organs and developmental stages. Our genomics analysis of maize MAPKKK genes provides important information for evolutionary and functional characterization of this family in maize.

  5. Genome-wide identification and analysis of expression profiles of maize mitogen-activated protein kinase kinase kinase.

    Directory of Open Access Journals (Sweden)

    Xiangpei Kong

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are highly conserved signal transduction model in animals, yeast and plants. Plant MAPK cascades have been implicated in development and stress responses. Although MAPKKKs have been investigated in several plant species including Arabidopsis and rice, no systematic analysis has been conducted in maize. In this study, we performed a bioinformatics analysis of the entire maize genome and identified 74 MAPKKK genes. Phylogenetic analyses of MAPKKKs from maize, rice and Arabidopsis have classified them into three subgroups, which included Raf, ZIK and MEKK. Evolutionary relationships within subfamilies were also supported by exon-intron organizations and the conserved protein motifs. Further expression analysis of the MAPKKKs in microarray databases revealed that MAPKKKs were involved in important signaling pathways in maize different organs and developmental stages. Our genomics analysis of maize MAPKKK genes provides important information for evolutionary and functional characterization of this family in maize.

  6. NFκB-inducing kinase inhibits NFκB activity specifically in neurons of the CNS.

    Science.gov (United States)

    Mao, Xianrong; Phanavanh, Bounleut; Hamdan, Hamdan; Moerman-Herzog, Andréa M; Barger, Steven W

    2016-04-01

    mechanisms, including the potential for a neuron-specific protein (e.g., one of the 14-3-3 class) to create a novel complex in neurons, whereas the C-terminus may interact directly with NFκB. [Structure of NIK is based on Liu J., Sudom A., Min X., Cao Z., Gao X., Ayres M., Lee F., Cao P., Johnstone S., Plotnikova O., Walker N., Chen G., and Wang Z. (2012) Structure of the nuclear factor κB-inducing kinase (NIK) kinase domain reveals a constitutively active conformation. J Biol Chem. 287, 27326-27334); N-terminal lobe is oriented at top]. PMID:26778773

  7. INHIBITING MAP KINASE ACTIVITY PREVENTS CALCIUM TRANSIENTS AND MITOSIS ENTRY IN EARLY SEA URCHIN EMBRYOS

    OpenAIRE

    Philipova, Rada; Larman, Mark G.; Leckie, Calum P.; Harrison, Patrick K.; Groigno, Laurence; Whitaker, Michael

    2005-01-01

    A transient calcium increase triggers nuclear envelope breakdown (mitosis entry) in sea urchin embryos. Cdk1/cyclin B kinase activation is also known to be required for mitosis entry. More recently MAP kinase activity has also been shown to increase during mitosis. In sea urchin embryos both kinases show a similar activation profile, peaking at the time of mitosis entry.

  8. DMPD: Receptor tyrosine kinases and the regulation of macrophage activation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14726496 Receptor tyrosine kinases and the regulation of macrophage activation. Cor...(.csml) Show Receptor tyrosine kinases and the regulation of macrophage activation. PubmedID 14726496 Title ...Receptor tyrosine kinases and the regulation of macrophage activation. Authors Co

  9. Regulation of AMP-activated protein kinase by LKB1 and CaMKK in adipocytes

    DEFF Research Database (Denmark)

    Gormand, Amélie; Henriksson, Emma; Ström, Kristoffer;

    2011-01-01

    AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates cellular and whole body energy homeostasis. In adipose tissue, activation of AMPK has been demonstrated in response to a variety of extracellular stimuli. However, the upstream kinase that activates AMPK in adipocytes...

  10. Activation and signaling of the p38 MAP kinase pathway

    Institute of Scientific and Technical Information of China (English)

    Tyler ZARUBIN; Jiahuai HAN

    2005-01-01

    The family members of the mitogen-activated protein (MAP) kinases mediate a wide variety of cellular behaviors in response to extracellular stimuli. One of the four main sub-groups, the p38 group of MAP kinases, serve as a nexus for signal transduction and play a vital role in numerous biological processes. In this review, we highlight the known characteristics and components of the p38 pathway along with the mechanism and consequences of p38 activation. We focus on the role of p38 as a signal transduction mediator and examine the evidence linking p38 to inflammation, cell cycle, cell death, development, cell differentiation, senescence and tumorigenesis in specific cell types. Upstream and downstream components of p38 are described and questions remaining to be answered are posed. Finally, we propose several directions for future research on p38.

  11. PRINT: A Protein Bioconjugation Method with Exquisite N-terminal Specificity

    Science.gov (United States)

    Sur, Surojit; Qiao, Yuan; Fries, Anja; O'Meally, Robert N.; Cole, Robert N.; Kinzler, Kenneth W.; Vogelstein, Bert; Zhou, Shibin

    2015-12-01

    Chemical conjugation is commonly used to enhance the pharmacokinetics, biodistribution, and potency of protein therapeutics, but often leads to non-specific modification or loss of bioactivity. Here, we present a simple, versatile and widely applicable method that allows exquisite N-terminal specific modification of proteins. Combining reversible side-chain blocking and protease mediated cleavage of a commonly used HIS tag appended to a protein, we generate with high yield and purity exquisitely site specific and selective bio-conjugates of TNF-α by using amine reactive NHS ester chemistry. We confirm the N terminal selectivity and specificity using mass spectral analyses and show near complete retention of the biological activity of our model protein both in vitro and in vivo murine models. We believe that this methodology would be applicable to a variety of potentially therapeutic proteins and the specificity afforded by this technique would allow for rapid generation of novel biologics.

  12. Polyphosphate Kinase from Activated Sludge Performing Enhanced Biological Phosphorus Removal†

    OpenAIRE

    Katherine D McMahon; Dojka, Michael A.; Pace, Norman R.; Jenkins, David; Keasling, Jay D.

    2002-01-01

    A novel polyphosphate kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in Rhodocyclus-like β-Proteobacteria kn...

  13. Training volume, androgen use and serum creatine kinase activity.

    OpenAIRE

    Häkkinen, K; Alén, M

    1989-01-01

    Serum creatine kinase (CK) activities were investigated in elite male strength athletes (n = 20) during normal weight training and bodybuilding training (one training session per day), during high volume strength training (two sessions per day) and during strength training (one session per day) with the use of high dose synthetic androgens (five athletes in each subgroup). The findings demonstrated that the increase in serum CK was highest in the subgroup using androgens. These results sugges...

  14. Expanding the Phenotype Associated with NAA10-Related N-Terminal Acetylation Deficiency.

    Science.gov (United States)

    Saunier, Chloé; Støve, Svein Isungset; Popp, Bernt; Gérard, Bénédicte; Blenski, Marina; AhMew, Nicholas; de Bie, Charlotte; Goldenberg, Paula; Isidor, Bertrand; Keren, Boris; Leheup, Bruno; Lampert, Laetitia; Mignot, Cyril; Tezcan, Kamer; Mancini, Grazia M S; Nava, Caroline; Wasserstein, Melissa; Bruel, Ange-Line; Thevenon, Julien; Masurel, Alice; Duffourd, Yannis; Kuentz, Paul; Huet, Frédéric; Rivière, Jean-Baptiste; van Slegtenhorst, Marjon; Faivre, Laurence; Piton, Amélie; Reis, André; Arnesen, Thomas; Thauvin-Robinet, Christel; Zweier, Christiane

    2016-08-01

    N-terminal acetylation is a common protein modification in eukaryotes associated with numerous cellular processes. Inherited mutations in NAA10, encoding the catalytic subunit of the major N-terminal acetylation complex NatA have been associated with diverse, syndromic X-linked recessive disorders, whereas de novo missense mutations have been reported in one male and one female individual with severe intellectual disability but otherwise unspecific phenotypes. Thus, the full genetic and clinical spectrum of NAA10 deficiency is yet to be delineated. We identified three different novel and one known missense mutation in NAA10, de novo in 11 females, and due to maternal germ line mosaicism in another girl and her more severely affected and deceased brother. In vitro enzymatic assays for the novel, recurrent mutations p.(Arg83Cys) and p.(Phe128Leu) revealed reduced catalytic activity. X-inactivation was random in five females. The core phenotype of X-linked NAA10-related N-terminal-acetyltransferase deficiency in both males and females includes developmental delay, severe intellectual disability, postnatal growth failure with severe microcephaly, and skeletal or cardiac anomalies. Genotype-phenotype correlations within and between both genders are complex and may include various factors such as location and nature of mutations, enzymatic stability and activity, and X-inactivation in females. PMID:27094817

  15. Structure of the intact ATM/Tel1 kinase

    Science.gov (United States)

    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.

  16. Neuroprotective effect of osthole against oxygen and glucose deprivation in rat cortical neurons: involvement of mitogen-activated protein kinase pathway.

    Science.gov (United States)

    Chen, T; Liu, W; Chao, X; Qu, Y; Zhang, L; Luo, P; Xie, K; Huo, J; Fei, Z

    2011-06-01

    Osthole, a bioactive simple coumarin derivative extracted from many medicinal plants such as Cnidium monnieri (L.) Cusson, exerts a broad spectrum of pharmacological activities and is considered to have potential therapeutic applications. The aim of this study was to investigate the potential neuroprotective role of osthole against ischemic injury in vitro, as well as the potential mechanism. Cultured cortical neurons were exposed to oxygen and glucose deprivation (OGD) for 4 h followed by a 24 h reperfusion. Osthole exhibited remarkable neuroprotection in a dose-dependent manner and the effect required presence of osthole during both OGD and reperfusion phases. Western blot was used to examine the activation of three members of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 kinase (p38). We found that osthole prolonged activation of ERK1/2 and prevented activation of JNK. Furthermore, we investigated the effects of MAPKs inhibitors on osthole-induced protection. The results demonstrated that the protection of osthole was partly reversed by PD98059, a selective inhibitor of ERK1/2, but further enhanced by the JNK inhibitor SP600125. In addition, osthole-induced reduction of neuronal apoptosis was abrogated by the ERK1/2 inhibitor PD98059, whereas the total neuronal death was further decreased by the JNK inhibitor SP600125. In summary, these data suggested that osthole had neuroprotective effect against ischemic injury in vitro, and the protection possibly was associated with prolonged activation of ERK1/2 and suppression of JNK activity. PMID:21453755

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

    DEFF Research Database (Denmark)

    Köpper, Frederik; Bierwirth, Cathrin; Schön, Margarete;

    2013-01-01

    knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation......DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed....... Moreover, MK2 activity was required for damage response, accumulation of ssDNA, and decreased survival when cells were treated with the nucleoside analogue gemcitabine or when the checkpoint kinase Chk1 was antagonized. By using DNA fiber assays, we found that MK2 inhibition or knockdown rescued DNA...

  18. Mutation of androgen receptor N-terminal phosphorylation site Tyr-267 leads to inhibition of nuclear translocation and DNA binding.

    Directory of Open Access Journals (Sweden)

    Mehmet Karaca

    Full Text Available Reactivation of androgen receptor (AR may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.

  19. Mutation of androgen receptor N-terminal phosphorylation site Tyr-267 leads to inhibition of nuclear translocation and DNA binding.

    Science.gov (United States)

    Karaca, Mehmet; Liu, Yuanbo; Zhang, Zhentao; De Silva, Dinuka; Parker, Joel S; Earp, H Shelton; Whang, Young E

    2015-01-01

    Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.

  20. Proteinase K processing of rabbit muscle creatine kinase

    DEFF Research Database (Denmark)

    Leydier, C; Andersen, Jens S.; Couthon, F;

    1997-01-01

    Proteinase K cleaves selectively both cytosolic and mitochondrial isoforms of creatine kinase leading to the appearance of two fragments, a large N-terminal one (K1) and a small C-terminal peptide (K2) which remain associated together. The loss of enzymatic activity correlates with the extent...... of monomer cleavage. N-terminal sequencing of the K2 fragments from rabbit cytosolic and pig mitochondrial creatine kinase shows that these peptides begin with A328 and A324, respectively. Electrospray ionization mass spectrometry demonstrates that K2 peptide is composed of 53 residues (A328-K380). However......, the C-terminal end of the K1 fragment is not A327 as expected, but D325. Thus, the amino acids residues T326 and A327 have been eliminated by the protease....

  1. Modulation of the protein kinase activity of mTOR.

    Science.gov (United States)

    Lawrence, J C; Lin, T A; McMahon, L P; Choi, K M

    2004-01-01

    mTOR is a founding member of a family of protein kinases having catalytic domains homologous to those in phosphatidylinositol 3-OH kinase. mTOR participates in the control by insulin of the phosphorylation of lipin, which is required for adipocyte differentiation, and the two translational regulators, p70S6K and PHAS-I. The phosphorylation of mTOR, itself, is stimulated by insulin in Ser2448, a site that is also phosphorylated by protein kinase B (PKB) in vitro and in response to activation of PKB activity in vivo. Ser2448 is located in a short stretch of amino acids not found in the two TOR proteins in yeast. A mutant mTOR lacking this stretch exhibited increased activity, and binding of the antibody, mTAb-1, to this region markedly increased mTOR activity. In contrast, rapamycin-FKBP12 inhibited mTOR activity towards both PHAS-I and p70S6K, although this complex inhibited the phosphorylation of some sites more than that of others. Mutating Ser2035 to Ile in the FKBP12-rapamycin binding domain rendered mTOR resistant to inhibition by rapamycin. Unexpectedly, this mutation markedly decreased the ability of mTOR to phosphorylate certain sites in both PHAS-I and p70S6K. The results support the hypotheses that rapamycin disrupts substrate recognition instead of directly inhibiting phosphotransferase activity and that mTOR activity in cells is controlled by the phosphorylation of an inhibitory regulatory domain containing the mTAb-1 epitope. PMID:14560959

  2. Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

    NARCIS (Netherlands)

    Parikh, Kaushal; Diks, Sander H.; Tuynman, Jurriaan H. B.; Verhaar, Auke; Lowenberg, Mark; Hommes, Daan W.; Joore, Jos; Pandey, Akhilesh; Peppelenbosch, Maikel P.

    2009-01-01

    Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to pred

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

    Directory of Open Access Journals (Sweden)

    Anshuman Dixit

    2009-08-01

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

  4. Mutations within the putative active site of heterodimeric deoxyguanosine kinase block the allosteric activation of the deoxyadenosine kinase subunit.

    Science.gov (United States)

    Park, Inshik; Ives, David H

    2002-03-31

    Replacement of the Asp-84 residue of the deoxyguanosine kinase subunit of the tandem deoxyadenosine kinase/ deoxyguanosine kinase (dAK/dGK) from Lactobacillus acidophilus R-26 by Ala, Asn, or Glu produced increased Km values for deoxyguanosine on dGK. However, it did not seem to affect the binding of Mg-ATP. The Asp-84 dGK replacements had no apparent effect on the binding of deoxyadenosine by dAK. However, the mutant dGKs were no longer inhibited by dGTP, normally a potent distal endproduct inhibitor of dGK. Moreover, the allosteric activation of dAK activity by dGTP or dGuo was lost in the modified heterodimeric dAK/dGK enzyme. Therefore, it seems very likely that Asp-84 participates in dGuo binding at the active site of the dGK subunit of dAK/dGK from Lactobacillus acidophilus R-26.

  5. Structure of the human histone chaperone FACT Spt16 N-terminal domain

    Energy Technology Data Exchange (ETDEWEB)

    Marcianò, G.; Huang, D. T., E-mail: d.huang@beatson.gla.ac.uk [Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, Scotland (United Kingdom)

    2016-01-22

    The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.

  6. Cellular reprogramming through mitogen-activated protein kinases

    Directory of Open Access Journals (Sweden)

    Justin eLee

    2015-10-01

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

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

    Directory of Open Access Journals (Sweden)

    Matthew J. Randall

    2013-01-01

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

  8. Activation of GABA(B) receptors inhibits protein kinase B/glycogen synthase kinase 3 signaling.

    Science.gov (United States)

    Lu, Frances Fangjia; Su, Ping; Liu, Fang; Daskalakis, Zafiris J

    2012-11-28

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

  9. A mathematical model of human thymidine kinase 2 activity

    DEFF Research Database (Denmark)

    Radivoyevitch, Tom; Munch-Petersen, Birgitte; Wang, Liya;

    2011-01-01

    _ The mitochondrial enzyme thymidine kinase 2 (TK2) phosphorylates deoxythymidine (dT) and deoxycytidine (dC) to form dTMP and dCMP, which in cells rapidly become the negative-feedback end-products dTTP and dCTP. TK2 kinetic activity exhibits Hill coefficients of ∼0.5 (apparent negative...... cooperativity) for dT and ∼1 for dC. We present a mathematical model of TK2 activity that is applicable if TK2 exists as two monomer forms in equilibrium....

  10. Activation of tracheal smooth muscle contraction: synergism between Ca2+ and activators of protein kinase C.

    OpenAIRE

    Park, S.; Rasmussen, H

    1985-01-01

    The effects of divalent ionophores (A23187 and ionomycin), Ca2+ channel agonist (BAY K 8644), and protein kinase C (C-kinase) activators [phorbol 12-myristate 13-acetate (PMA), mezerein] on bovine tracheal smooth muscle contraction were investigated. A23187 (5 microM) and ionomycin (0.5 microM) produced a prompt but transient contraction. C-kinase activators either produced no effect--e.g., PMA at 200 nM--or produced a rise in tension that was slow in onset but then gradually increased--e.g.,...

  11. Regulation of protein kinase B/Akt activity and Ser473 phosphorylation by protein kinase Calpha in endothelial cells.

    Science.gov (United States)

    Partovian, Chohreh; Simons, Michael

    2004-08-01

    Protein kinase Balpha (PKBalpha/Akt-1) is a key mediator of multiple signaling pathways involved in angiogenesis, cell proliferation and apoptosis among others. The unphosphorylated form of Akt-1 is virtually inactive and its full activation requires two phosphatidylinositol-3,4,5-triphosphate-dependent phosphorylation events, Thr308 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser473 by an undefined kinase that has been termed PDK2. Recent studies have suggested that the Ser473 kinase is a plasma membrane raft-associated kinase. In this study we show that protein kinase Calpha (PKCalpha) translocates to the membrane rafts in response to insulin growth factor-1 (IGF-1) stimulation. Overexpression of PKCalpha increases Ser473 phosphorylation and Akt-1 activity, while inhibition of its activity or expression decreases IGF-1-dependent activation of Akt-1. Furthermore, in vitro, in the presence of phospholipids and calcium, PKCalpha directly phosphorylates Akt-1 at the Ser473 site. We conclude, therefore, that PKCalpha regulates Akt-1 activity via Ser473 phosphorylation and may function as PDK2 in endothelial cells. PMID:15157674

  12. Development of disposable RI device for diagnostic LOC using kinase activity measurement

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seok Jae; Ahn, Chi Won; Kim, Hee Yeon; Kim, Byeong Il; Park, Jae Hong; Kim, Kyung Min [KAIST, Daejeon (Korea, Republic of)

    2010-05-15

    A pratical method to determine the activity of kinase will be developed. The method will be used to construct a miniaturized analysis for the determination of kinase activity based on RI system. We fabricate miniaturized kinase assay system in a COC (Cyclo Olefin Copolymer) microfluidic chip. The demonstrated strategy demonstrates the usefulness of the microchips for performing enzymatic assays for which leads to multitude of kinase reaction simultaneously

  13. β-Catenin Binds to the Activation Function 2 Region of the Androgen Receptor and Modulates the Effects of the N-Terminal Domain and TIF2 on Ligand-Dependent Transcription

    OpenAIRE

    Song, Liang-Nian; Herrell, Roger; Byers, Stephen; Shah, Salimuddin; Wilson, Elizabeth M.; Gelmann, Edward P.

    2003-01-01

    β-Catenin is a multifunctional molecule that is activated by signaling through WNT receptors. β-Catenin can also enhance the transcriptional activity of some steroid hormone receptors such as the androgen receptor and retinoic acid receptor α. Androgens can affect nuclear translocation of β-catenin and influence its subcellular distribution. Using mammalian two-hybrid binding assays, analysis of reporter gene transcription, and coimmunoprecipitation, we now show that β-catenin binds to the an...

  14. P21-activated kinase 1 and breast cancer

    Institute of Scientific and Technical Information of China (English)

    Jun-Xiang Zhang; Da-Qiang Li; Rakesh Kumar

    2010-01-01

    @@ The p21 activated kinase 1 (PAK1) belongs to PAKs family, a group of highly evolutionarily conserved protein family of serine/threonine kinases, which acts as a downstream effector of the small GTPases Cdc42 and Rac1, firstly reported in 1994[1]. As a serine/threonine kinase, PAK1 plays an important role in many cellular functions including cell morphogenesis, motility, survival, mitosis, angiogenesis, and tumorigenesis. More than 40 proteins have been reported to be phosphorylated by PAK1[2]. Accumulating experimental data in multiple experimental systems provide compelling evidence that PAK1 plays an important role in breast cancer promotion and progression. PAK1 is overexpressed and/or hyperactivated in more than 50% of breast cancers[3]. On the other hand, PAK1 overexpression in estrogen receptor alpha (ER α) positive breast cancer is also closely associated with a reduced responsiveness to tamoxifen therapy[4]. Since PAK1 plays such a vital role in breast cancer, PAK1 targeted therapeutic approaches are likely to be useful in breast cancer treatment as well as in other human cancers with PAK1 upregulation and/or hyperactivation[5].

  15. Effects of protein kinase C activators and staurosporine on protein kinase activity, cell survival, and proliferation in Tetrahymena thermophila

    DEFF Research Database (Denmark)

    Straarup, EM; Schousboe, P; Hansen, HQ;

    1997-01-01

    with either PMA or OAG, or at 2,500 cells ml-1. At 500 cells ml-1 PMA induced the in vivo phosphorylation of at least six proteins. The myelin basic protein fragment 4-14 was phosphorylated in vitro in crude extracts of a culture of 250,000 cells ml-1. Both the in vivo and the in vitro phosphorylation were......Autocrine factors prevent cell death in the ciliate Tetrahymena thermophila, a unicellular eukaryote, in a chemically defined medium. At certain growth conditions these factors are released at a sufficient concentration by > 500 cells ml-1 to support cell survival and proliferation. The protein...... kinase C activators phorbol 12-myristate 13-acetate (PMA) or 1-oleyl 2-acetate glycerol (OAG) when added to 250 cells ml-1 supported cell survival and proliferation. In the presence of the serine and threonine kinase inhibitor staurosporine the cells died both at 250 cells ml-1 in cultures supplemented...

  16. Activation of the Antiviral Kinase PKR and Viral Countermeasures

    Directory of Open Access Journals (Sweden)

    Bianca Dauber

    2009-10-01

    Full Text Available The interferon-induced double-stranded (dsRNA-dependent protein kinase (PKR limits viral replication by an eIF2α-mediated block of translation. Although many negative-strand RNA viruses activate PKR, the responsible RNAs have long remained elusive, as dsRNA, the canonical activator of PKR, has not been detected in cells infected with such viruses. In this review we focus on the activating RNA molecules of different virus families, in particular the negative-strand RNA viruses. We discuss the recently identified non-canonical activators 5’-triphosphate RNA and the vRNP of influenza virus and give an update on strategies of selected RNA and DNA viruses to prevent activation of PKR.

  17. Kinase Signaling in Apoptosis Induced by Saturated Fatty Acids in Pancreatic β-Cells.

    Science.gov (United States)

    Šrámek, Jan; Němcová-Fürstová, Vlasta; Kovář, Jan

    2016-01-01

    Pancreatic β-cell failure and death is considered to be one of the main factors responsible for type 2 diabetes. It is caused by, in addition to hyperglycemia, chronic exposure to increased concentrations of fatty acids, mainly saturated fatty acids. Molecular mechanisms of apoptosis induction by saturated fatty acids in β-cells are not completely clear. It has been proposed that kinase signaling could be involved, particularly, c-Jun N-terminal kinase (JNK), protein kinase C (PKC), p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), and Akt kinases and their pathways. In this review, we discuss these kinases and their signaling pathways with respect to their possible role in apoptosis induction by saturated fatty acids in pancreatic β-cells. PMID:27626409

  18. Kinase Signaling in Apoptosis Induced by Saturated Fatty Acids in Pancreatic β-Cells

    Directory of Open Access Journals (Sweden)

    Jan Šrámek

    2016-09-01

    Full Text Available Pancreatic β-cell failure and death is considered to be one of the main factors responsible for type 2 diabetes. It is caused by, in addition to hyperglycemia, chronic exposure to increased concentrations of fatty acids, mainly saturated fatty acids. Molecular mechanisms of apoptosis induction by saturated fatty acids in β-cells are not completely clear. It has been proposed that kinase signaling could be involved, particularly, c-Jun N-terminal kinase (JNK, protein kinase C (PKC, p38 mitogen-activated protein kinase (p38 MAPK, extracellular signal-regulated kinase (ERK, and Akt kinases and their pathways. In this review, we discuss these kinases and their signaling pathways with respect to their possible role in apoptosis induction by saturated fatty acids in pancreatic β-cells.

  19. Structures of Rhodopsin Kinase in Different Ligand States Reveal Key Elements Involved in G Protein-coupled Receptor Kinase Activation

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Puja; Wang, Benlian; Maeda, Tadao; Palczewski, Krzysztof; Tesmer, John J.G. (Case Western); (Michigan)

    2008-10-08

    G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated heptahelical receptors, leading to their uncoupling from G proteins. Here we report six crystal structures of rhodopsin kinase (GRK1), revealing not only three distinct nucleotide-binding states of a GRK but also two key structural elements believed to be involved in the recognition of activated GPCRs. The first is the C-terminal extension of the kinase domain, which was observed in all nucleotide-bound GRK1 structures. The second is residues 5-30 of the N terminus, observed in one of the GRK1{center_dot}(Mg{sup 2+}){sub 2} {center_dot}ATP structures. The N terminus was also clearly phosphorylated, leading to the identification of two novel phosphorylation sites by mass spectral analysis. Co-localization of the N terminus and the C-terminal extension near the hinge of the kinase domain suggests that activated GPCRs stimulate kinase activity by binding to this region to facilitate full closure of the kinase domain.

  20. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

    KAUST Repository

    Zourelidou, Melina

    2014-06-19

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  1. Subtype activation and interaction of protein kinase C and mitogen-activated protein kinase controlling receptor expression in cerebral arteries and microvessels after subarachnoid hemorrhage

    DEFF Research Database (Denmark)

    Ansar, S.; Edvinsson, L.

    2008-01-01

    ischemia after SAH in cerebral arteries and microvessels and to examine temporal activation of the kinases. We hypothesize that treatment with a MAPK or PKC inhibitor will prevent the SAH-induced kinase activation in brain vessels. METHODS: SAH was induced by injecting 250 microL blood...

  2. Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Deficiency Reduces Insulin Sensitivity in High-Fat Diet-Fed Mice

    NARCIS (Netherlands)

    de Boer, Jan Freark; Dikkers, Arne; Jurdzinski, Angelika; von Felden, Johann; Gaestel, Matthias; Bavendiek, Udo; Tietge, Uwe J. F.

    2014-01-01

    Adipose tissue inflammation is considered an important contributor to insulin resistance. Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a major downstream target of p38 MAPK and enhances inflammatory processes. In line with the role of MK2 as contributor to inflammation, MK2(-

  3. Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells

    DEFF Research Database (Denmark)

    Frödin, M; Peraldi, P; Van Obberghen, E

    1994-01-01

    Mitogen-activated protein (MAP) kinases are activated in response to a large variety of extracellular signals, including growth factors, hormones, and neurotransmitters, which activate distinct intracellular signaling pathways. Their activation by the cAMP-dependent pathway, however, has not been...

  4. Substitution of lysine for arginine in the N-terminal 217th amino acid residue of the H gamma II of Staphylococcal gamma-hemolysin lowers the activity of the toxin.

    Science.gov (United States)

    Sudo, K; Choorit, W; Asami, I; Kaneko, J; Muramoto, K; Kamio, Y

    1995-09-01

    The staphylococcal toxin gamma-hemolysin consists of two protein components, LukF and H gamma II. Staphylococcus aureus P83 was found to have five components, LukF, LukF-PV, LukM, LukS, and H gamma II for leukocidin or gamma-hemolysin. H gamma II of S. aureus P83 was demonstrated to be a naturally-occurring analogous molecule of H gamma II [H gamma II(P83)], in which the 217th arginine residue was replaced by lysine. The H gamma II(P83) showed about 50% of the hemolytic activity of normal H gamma II in the presence of LukF.

  5. Pyruvate dehydrogenase kinase-4 structures reveal a metastable open conformation fostering robust core-free basal activity.

    Science.gov (United States)

    Wynn, R Max; Kato, Masato; Chuang, Jacinta L; Tso, Shih-Chia; Li, Jun; Chuang, David T

    2008-09-12

    Human pyruvate dehydrogenase complex (PDC) is down-regulated by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. PDK4 is overexpressed in skeletal muscle in type 2 diabetes, resulting in impaired glucose utilization. Here we show that human PDK4 has robust core-free basal activity, which is considerably higher than activity levels of other PDK isoforms stimulated by the PDC core. PDK4 binds the L3 lipoyl domain, but its activity is not significantly stimulated by any individual lipoyl domains or the core of PDC. The 2.0-A crystal structures of the PDK4 dimer with bound ADP reveal an open conformation with a wider active-site cleft, compared with that in the closed conformation epitomized by the PDK2-ADP structure. The open conformation in PDK4 shows partially ordered C-terminal cross-tails, in which the conserved DW (Asp(394)-Trp(395)) motif from one subunit anchors to the N-terminal domain of the other subunit. The open conformation fosters a reduced binding affinity for ADP, facilitating the efficient removal of product inhibition by this nucleotide. Alteration or deletion of the DW-motif disrupts the C-terminal cross-tail anchor, resulting in the closed conformation and the nearly complete inactivation of PDK4. Fluorescence quenching and enzyme activity data suggest that compounds AZD7545 and dichloroacetate lock PDK4 in the open and the closed conformational states, respectively. We propose that PDK4 with bound ADP exists in equilibrium between the open and the closed conformations. The favored metastable open conformation is responsible for the robust basal activity of PDK4 in the absence of the PDC core. PMID:18658136

  6. Directed evolution of the TALE N-terminal domain for recognition of all 5' bases.

    Science.gov (United States)

    Lamb, Brian M; Mercer, Andrew C; Barbas, Carlos F

    2013-11-01

    Transcription activator-like effector (TALE) proteins can be designed to bind virtually any DNA sequence. General guidelines for design of TALE DNA-binding domains suggest that the 5'-most base of the DNA sequence bound by the TALE (the N0 base) should be a thymine. We quantified the N0 requirement by analysis of the activities of TALE transcription factors (TALE-TF), TALE recombinases (TALE-R) and TALE nucleases (TALENs) with each DNA base at this position. In the absence of a 5' T, we observed decreases in TALE activity up to >1000-fold in TALE-TF activity, up to 100-fold in TALE-R activity and up to 10-fold reduction in TALEN activity compared with target sequences containing a 5' T. To develop TALE architectures that recognize all possible N0 bases, we used structure-guided library design coupled with TALE-R activity selections to evolve novel TALE N-terminal domains to accommodate any N0 base. A G-selective domain and broadly reactive domains were isolated and characterized. The engineered TALE domains selected in the TALE-R format demonstrated modularity and were active in TALE-TF and TALEN architectures. Evolved N-terminal domains provide effective and unconstrained TALE-based targeting of any DNA sequence as TALE binding proteins and designer enzymes.

  7. Subtype activation and interaction of protein kinase C and mitogen-activated protein kinase controlling receptor expression in cerebral arteries and microvessels after subarachnoid hemorrhage

    DEFF Research Database (Denmark)

    Ansar, Saema; Edvinsson, Lars

    2008-01-01

    BACKGROUND AND PURPOSE: The pathogenesis of cerebral ischemia associated with subarachnoid hemorrhage (SAH) still remains elusive. The aim of this study was to examine the involvement of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) subtypes in the pathophysiology of cerebral...... enhanced phosphorylation only at 48 hours after SAH. The pattern was identical in large cerebral arteries and in intracerebral microvessels. Treatment with either the PKC (RO-31-7549) or the raf (SB386023-b) inhibitor prevented the kinase activation. CONCLUSIONS: The results show that specific subtypes...... ischemia after SAH in cerebral arteries and microvessels and to examine temporal activation of the kinases. We hypothesize that treatment with a MAPK or PKC inhibitor will prevent the SAH-induced kinase activation in brain vessels. METHODS: SAH was induced by injecting 250 microL blood...

  8. Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

    Directory of Open Access Journals (Sweden)

    Belén Mezquita

    2014-02-01

    Full Text Available One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT and a truncated isoform of VEGFR-1 (i21-VEGFR-1, which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer.

  9. N-terminal processing of membrane-targeted MnSOD and formation of multiple active superoxide dismutase dimers in the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC7120.

    Science.gov (United States)

    Raghavan, Prashanth S; Rajaram, Hema; Apte, Shree K

    2013-10-01

    Anabaena sp. strain PCC7120 expresses a 30 kDa manganese-dependent superoxide dismutase (MnSOD) comprising a hydrophobic region (signal peptide + linker peptide) attached to a catalytic unit. Bioinformatics predicted cleavage of the signal peptide at (25)CQPQ by signal peptidase and of the linker peptide by an Arg-C-like protease at the Arg52/Arg59 residue. The three predicted forms of MnSOD were immunodetected in Anabaena, with the 30 kDa MnSOD found exclusively in the membrane and the shorter 27 and 24 kDa forms found both in the membrane and soluble fractions. The corresponding sodA gene was truncated for (a) the first eight residues, or, (b) the signal peptide, or (c) the entire hydrophobic region, or (d) the Arg52/Arg59 residues were modified to serine. Overexpression of these MnSOD variants in recombinant Anabaena strains revealed that (a) the 30 kDa membrane-targeted MnSOD was cleaved by membrane-localized signal peptidase either during or after its transport through the membrane to release the 27 kDa form, either in the cytosol or in the periplasmic/thylakoid lumen, (b) the 27 kDa form was further cleaved to the 24 kDa form by Arg-C-like protease, both in the cytosol and in the periplasmic/thylakoid lumen, (c) deletion of signal peptide localized the MnSOD forms in the cytosol, and (d) alteration of the signal/linker peptide cleavage sites interfered with MnSOD localization and processing. Homo/heterodimerization of the 24 and 27 kDa forms of MnSOD and the cytosolic iron-dependent SOD results in multiple SOD activities, from a single MnSOD gene (sodA), in different cellular compartments of Anabaena.

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

    Science.gov (United States)

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

    2016-04-01

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

  11. CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity

    Science.gov (United States)

    Calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases but we made the surprising observation that soybean CDPK' and several Arabidopsis isoforms (AtCPK4 and AtCPK34) could also autophosphorylate on tyrosine residues. In studies with His6-GmCDPK', we ide...

  12. Linked decreases in Liver Kinase B1 and AMP-activated protein kinase activity modulate matrix catabolic responses to biomechanical injury in chondrocytes

    OpenAIRE

    Petursson, Freyr; Husa, Matt; June, Ron; Lotz, Martin; Terkeltaub, Robert; Liu-Bryan, Ru

    2013-01-01

    Abstract Introduction AMP-activated protein kinase (AMPK) maintains cultured chondrocyte matrix homeostasis in response to inflammatory cytokines. AMPK activity is decreased in human knee osteoarthritis (OA) chondrocytes. Liver kinase B1 (LKB1) is one of the upstream activators of AMPK. Hence, we examined the relationship between LKB1 and AMPK activity in OA and aging cartilages, and in chondrocytes subjected to inflammatory cytokine treatment and biomechanical compression injury, and p...

  13. Mitogen-activated protein kinase kinase activity is required for the G2/M transition of the cell cycle in mammalian fibroblasts

    OpenAIRE

    Wright, Jocelyn H.; Munar, Erlynda; Jameson, Damon R; Andreassen, Paul R.; Margolis, Robert L.; Seger, Rony; Krebs, Edwin G.

    1999-01-01

    The mitogen-activated protein kinase (MAPK) cascade is required for mitogenesis in somatic mammalian cells and is activated by a wide variety of oncogenic stimuli. Specific roles for this signaling module in growth were dissected by inhibiting MAPK kinase 1 (MAPKK1) activity in highly synchronized NIH 3T3 cells. In addition to the known role of this kinase in cell-cycle entry from G0, the level of MAPKK activity was observed to affect the kinetics of progression through both the G1 and G2 pha...

  14. Oscillatory change of SR-protein kinase activities during oocyte maturation meiosis in fish

    Institute of Scientific and Technical Information of China (English)

    杨仲安; 曹丹; 桂建芳

    2000-01-01

    The SR-protein kinase activity was analyzed and the cytological changes were observed during oocyte maturation in bisexual transparent color crucian carp ( Carassius auratus color variety). The results revealed that the SR-protein kinase activity was sensitive to the artificially induced spawning hormones, and the change of oscillatory activity was similar to that of the maturation-promoting factor (MPF) kinase that regulates meiotic cell cycle in fish.

  15. High quality, small molecule-activity datasets for kinase research [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Rajan Sharma

    2016-06-01

    Full Text Available Kinases regulate cell growth, movement, and death. Deregulated kinase activity is a frequent cause of disease. The therapeutic potential of kinase inhibitors has led to large amounts of published structure activity relationship (SAR data. Bioactivity databases such as the Kinase Knowledgebase (KKB, WOMBAT, GOSTAR, and ChEMBL provide researchers with quantitative data characterizing the activity of compounds across many biological assays. The KKB, for example, contains over 1.8M kinase structure-activity data points reported in peer-reviewed journals and patents. In the spirit of fostering methods development and validation worldwide, we have extracted and have made available from the KKB 258K structure activity data points and 76K associated unique chemical structures across eight kinase targets. These data are freely available for download within this data note.

  16. Regulation of tomato Prf by Pto-like protein kinases.

    Science.gov (United States)

    Mucyn, Tatiana S; Wu, Ai-Jiuan; Balmuth, Alexi L; Arasteh, Julia Maryam; Rathjen, John P

    2009-04-01

    Tomato Prf encodes a nucleotide-binding domain shared by Apaf-1, certain R proteins, and CED-4 fused to C-terminal leucine-rich repeats (NBARC-LRR) protein that is required for bacterial immunity to Pseudomonas syringae and sensitivity to the organophosphate fenthion. The signaling pathways involve two highly related protein kinases. Pto kinase mediates direct recognition of the bacterial effector proteins AvrPto or AvrPtoB. Fen kinase is required for fenthion sensitivity and recognition of bacterial effectors related to AvrPtoB. The role of Pto and its association with Prf has been characterized but Fen is poorly described. We show that, similar to Pto, Fen requires N-myristoylation and kinase activity for signaling and interacts with the N-terminal domain of Prf. Thus, the mechanisms of activation of Prf by the respective protein kinases are similar. Prf-Fen interaction is underlined by coregulatory mechanisms in which Prf negatively regulates Fen, most likely by controlling kinase activity. We further characterized negative regulation of Prf by Pto, and show that regulation is mediated by the previously described negative regulatory patch. Remarkably, the effectors released negative regulation of Prf in a manner dependent on Pto kinase activity. The data suggest a model in which Prf associates generally with Pto-like kinases in tightly regulated complexes, which are activated by effector-mediated disruption of negative regulation. Release of negative regulation may be a general feature of activation of NBARC-LRR proteins by cognate effectors.

  17. Amygdala kindling alters protein kinase C activity in dentate gyrus.

    Science.gov (United States)

    Chen, S J; Desai, M A; Klann, E; Winder, D G; Sweatt, J D; Conn, P J

    1992-11-01

    Kindling is a use-dependent form of synaptic plasticity and a widely used model of epilepsy. Although kindling has been widely studied, the molecular mechanisms underlying induction of this phenomenon are not well understood. We determined the effect of amygdala kindling on protein kinase C (PKC) activity in various regions of rat brain. Kindling stimulation markedly elevated basal (Ca(2+)-independent) and Ca(2+)-stimulated phosphorylation of an endogenous PKC substrate (which we have termed P17) in homogenates of dentate gyrus, assayed 2 h after kindling stimulation. The increase in P17 phosphorylation appeared to be due at least in part to persistent PKC activation, as basal PKC activity assayed in vitro using an exogenous peptide substrate was increased in kindled dentate gyrus 2 h after the last kindling stimulation. A similar increase in basal PKC activity was observed in dentate gyrus 2 h after the first kindling stimulation. These results document a kindling-associated persistent PKC activation and suggest that the increased activity of PKC could play a role in the induction of the kindling effect.

  18. Molecular mechanism by which AMP-activated protein kinase activation promotes glycogen accumulation in muscle

    DEFF Research Database (Denmark)

    Hunter, Roger W; Treebak, Jonas Thue; Wojtaszewski, Jørgen;

    2011-01-01

    OBJECTIVE During energy stress, AMP-activated protein kinase (AMPK) promotes glucose transport and glycolysis for ATP production, while it is thought to inhibit anabolic glycogen synthesis by suppressing the activity of glycogen synthase (GS) to maintain the energy balance in muscle. Paradoxicall...... and subsequent rise in cellular [G6P]....

  19. Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.

    Directory of Open Access Journals (Sweden)

    Boris Zybailov

    Full Text Available Characterization of the chloroplast proteome is needed to understand the essential contribution of the chloroplast to plant growth and development. Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. Novel proteins include various kinases and putative nucleotide binding proteins. Based on repeated and independent MS based protein identifications requiring multiple matched peptide sequences, as well as literature, 916 nuclear-encoded proteins were assigned with high confidence to the plastid, of which 86% had a predicted chloroplast transit peptide (cTP. The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude. Comparison to gel-based quantification demonstrates that 'spectral counting' can provide large scale protein quantification for Arabidopsis. This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. The abundance data for 550 stromal proteins was used to understand abundance of metabolic pathways and chloroplast processes. We highlight the abundance of 48 stromal proteins involved in post-translational proteome homeostasis (including aminopeptidases, proteases, deformylases, chaperones, protein sorting components and discuss the biological implications. N-terminal modifications were identified for a subset of nuclear- and chloroplast-encoded proteins and a novel N-terminal acetylation motif was discovered. Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction. No evidence

  20. Polo-Like Kinase-1 Controls Aurora A Destruction by Activating APC/C-Cdh1

    NARCIS (Netherlands)

    van Leuken, Renske; Clijsters, Linda; van Zon, Wouter; Lim, Dan; Yao, XueBiao; Wolthuis, Rob M. F.; Yaffe, Michael B.; Medema, Rene H.; van Vugt, Marcel A. T. M.

    2009-01-01

    Polo-like kinase-1 (Plk1) is activated before mitosis by Aurora A and its cofactor Bora. In mitosis, Bora is degraded in a manner dependent on Plk1 kinase activity and the E3 ubiquitin ligase SCF-beta TrCP. Here, we show that Plk1 is also required for the timely destruction of its activator Aurora A

  1. Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the γ1 subunit

    Science.gov (United States)

    Wu, Chongming; Guo, Yanshen; Su, Yan; Zhang, Xue; Luan, Hong; Zhang, Xiaopo; Zhu, Huixin; He, Huixia; Wang, Xiaoliang; Sun, Guibo; Sun, Xiaobo; Guo, Peng; Zhu, Ping

    2014-01-01

    Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP-activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin-induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)-elicited intracellular lipid accumulation and increased AMPK activity in a dose-dependent manner. Cordycepin-induced AMPK activation was not accompanied by changes in either the intracellular levels of AMP or the AMP/ATP ratio, nor was it influenced by calmodulin-dependent protein kinase kinase (CaMKK) inhibition; however, this activation was significantly suppressed by liver kinase B1 (LKB1) knockdown. Molecular docking, fluorescent and circular dichroism measurements showed that cordycepin interacted with the γ1 subunit of AMPK. Knockdown of AMPKγ1 by siRNA substantially abolished the effects of cordycepin on AMPK activation and lipid regulation. The modulating effects of cordycepin on the mRNA levels of key lipid regulatory genes were also largely reversed when AMPKγ1 expression was inhibited. Together, these data suggest that cordycepin may inhibit intracellular lipid accumulation through activation of AMPK via interaction with the γ1 subunit. PMID:24286368

  2. H pylori stimulates proliferation of gastric cancer cells through activating mitogen-activated protein kinase cascade

    Institute of Scientific and Technical Information of China (English)

    Yong-Chang Chen; Ying Wang; Jing-Yan Li; Wen-Rong Xu; You-Li Zhang

    2006-01-01

    AIM: To explore the mechanism by which H pylori causes activation of gastric epithelial cells.METHODS: A VacA (+) and CagA (+) standard Hpyloriline NCTC 11637 and a human gastric adenocarcinoma derived gastric epithelial cell line BGC-823 were applied in the study. MTT assay and 3H-TdR incorporation test were used to detect the proliferation of BGC-823 cells and Western blotting was used to detect the activity and existence of related proteins.RESULTS: Incubation with Hpylori extract increased the proliferation of gastric epithelial cells, reflected by both live cell number and DNA synthesis rate. The activity of extracellular signal-regulated protein kinase (ERK) signal transduction cascade increased within 20 min after incubation with Hpylori extract and appeared to be a sustained event. MAPK/ERK kinase (MEK) inhibitor PD98059abolished the action of H pylori extract on both ERK activity and cell proliferation. Incubation with H pyloriextract increased c-Fos expression and SRE-dependentgene expression. H pylori extract caused phosphorylation of several proteins including a protein with molecular size of 97.4 kDa and tyrosine kinase inhibitor genistein inhibited the activation of ERK and the proliferation of cells caused by H pylori extract.CONCLUSION: Biologically active elements in H pylori extract cause proliferation of gastric epithelial cells through activating tyrosine kinase and ERK signal transduction cascade.

  3. Estrus cycle effect on muscle tyrosine kinase activity in bitches.

    Science.gov (United States)

    Gomes Pöppl, Álan; Costa Valle, Sandra; Hilário Díaz González, Félix; de Castro Beck, Carlos Afonso; Kucharski, Luiz Carlos; Silveira Martins Da Silva, Roselis

    2012-03-01

    Estrus cycle is a well recognized cause of insulin resistance in bitches. The insulin receptor (IR) as well as the insulin-like growth factor-I receptor belong to the same subfamily of tyrosine kinase (TK) receptors. The objective of this study was to evaluate basal TK activity in muscle tissue of bitches during the estrus cycle. Twenty-four bitches were used in the study (7 in anestrus, 7 in estrus, and 10 in diestrus). Muscle samples, taken after spaying surgery to determine TK activity, were immediately frozen in liquid nitrogen and then stored at -80°C until the membranes were prepared by sequential centrifugation after being homogenized. TK activity was determined by Poly (Glu 4:Tyr 1) phosphorylation and expressed in cpm/μg of protein. TK activity was significantly lower (P < 0.001) in the animals in estrus (104.5 ± 11.9 cpm/μg of protein) and diestrus (94.5 ± 16.9 cpm/μg of protein) when compared with bitches in anestrus (183.2 ± 39.2 cpm/μg of protein). These results demonstrate, for the first time, lower basal TK activity in the muscle tissue of female dogs during estrus and diestrus, which may represent lower insulin signaling capacity, opening a new field of investigation into the molecular mechanisms of insulin resistance in dogs. PMID:22139063

  4. Parkin Regulates the Activity of Pyruvate Kinase M2.

    Science.gov (United States)

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

    2016-05-01

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

  5. Functional characterization of a special thermophilic multifunctional amylase OPMA-N and its N-terminal domain

    Institute of Scientific and Technical Information of China (English)

    Fan Li; Xuejun Zhu; Yanfei Li; Hao Cao; Yingjiu Zhang

    2011-01-01

    A gene encoding a special thermophilic muitifunetional amylase OPMA-N was cloned from Bacillus sp. ZW25311. OPMA-N has an additional 124-residue N-terminal domain compared with typical amylases and forms a relatively independent domain with a IS-pleated sheet and random coil structure. Here we reported an unusual substrate and product specificities of OPMA-N and the impact of the additional N-terminal domain (1-124 aa) on the function and properties of OPMA-N. Both OPMAN (12.82 U/mg) and its N-terminal domain-truncated AOPMA-N (12.55 U/mg) only degraded starch to produce oligosaccharides including maltose, maltotriose, isomaitotriose, and isomaitotetraose, but not to produce glucose. Therefore, the N-terminal domain did not determine its substrate and product specificities that were probably regulated by its C-terminal IS-pleated sheet structure. However, the N-terminal domain of OPMA-N seemed to modulate its catalytic feature, leading to the production of more isomaitotriose and less maltose, and it seemed to contribute to OPMA-N's thermostability since OPMA-N showed higher activity than AOPMA-N in a temperature range from 40 to 80~C and the halflife (tl) was 5 h for OPMA-N and 2 h for AOPMA-N at 60~C. Both OPMA-N and AOPMA-N were Ca-independent, but their activities could be influenced by Cu2+, Niz+, Zn2+, EDTA, SDS (1 mM), or Triton-X100 (1%). Kinetic analysis and starch-adsorption assay indicated that the N-terminal domain of OPMA-N could increase the OPMA-N-starch binding and subsequently increase the catalytic efficiency of OPMA-N for starch. In particular, the N-terminal domain of OPMA-N did not determine its oligomerization, because both OPMA-N and AOPMA-N could exist in the forms of monomer, homodimer, and homooligomer at the same time.

  6. Predicting Kinase Activity in Angiotensin Receptor Phosphoproteomes Based on Sequence-Motifs and Interactions

    DEFF Research Database (Denmark)

    Bøgebo, Rikke; Horn, Heiko; Olsen, Jesper V;

    2014-01-01

    -arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data...... developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets....

  7. UV ACTIVATION OF RECEPTOR TYROSINE KINASE-ACTIVITY

    NARCIS (Netherlands)

    COFFER, PJ; BURGERING, BMT; PEPPELENBOSCH, MP; BOS, JL; KRUIJER, W

    1995-01-01

    The exposure of mammalian cells to ultraviolet radiation (UV) may lead to DNA damage resulting in mutation and thus possibly cancer, while irradiation can further act as a potent tumour promoter. In addition UV induces p21ras-mediated signalling leading to activation of transcription factors such as

  8. Regulation of Akt/PKB activity by P21-activated kinase in cardiomyocytes.

    Science.gov (United States)

    Mao, Kai; Kobayashi, Satoru; Jaffer, Zahara M; Huang, Yuan; Volden, Paul; Chernoff, Jonathan; Liang, Qiangrong

    2008-02-01

    Akt/PKB is a critical regulator of cardiac function and morphology, and its activity is governed by dual phosphorylation at active loop (Thr308) by phosphoinositide-dependent protein kinase-1 (PDK1) and at carboxyl-terminal hydrophobic motif (Ser473) by a putative PDK2. P21-activated kinase-1 (Pak1) is a serine/threonine protein kinase implicated in the regulation of cardiac hypertrophy and contractility and was shown previously to activate Akt through an undefined mechanism. Here we report Pak1 as a potential PDK2 that is essential for Akt activity in cardiomyocytes. Both Pak1 and Akt can be activated by multiple hypertrophic stimuli or growth factors in a phosphatidylinositol-3-kinase (PI3K)-dependent manner. Pak1 overexpression induces Akt phosphorylation at both Ser473 and Thr308 in cardiomyocytes. Conversely, silencing or inactivating Pak1 gene diminishes Akt phosphorylation in vitro and in vivo. Purified Pak1 can directly phosphorylate Akt only at Ser473, suggesting that Pak1 may be a relevant PDK2 responsible for AKT Ser473 phosphorylation in cardiomyocytes. In addition, Pak1 protects cardiomyocytes from cell death, which is blocked by Akt inhibition. Our results connect two important regulators of cellular physiological functions and provide a potential mechanism for Pak1 signaling in cardiomyocytes. PMID:18054038

  9. Cdc7p-Dbf4p regulates mitotic exit by inhibiting Polo kinase.

    Directory of Open Access Journals (Sweden)

    Charles T Miller

    2009-05-01

    Full Text Available Cdc7p-Dbf4p is a conserved protein kinase required for the initiation of DNA replication. The Dbf4p regulatory subunit binds Cdc7p and is essential for Cdc7p kinase activation, however, the N-terminal third of Dbf4p is dispensable for its essential replication activities. Here, we define a short N-terminal Dbf4p region that targets Cdc7p-Dbf4p kinase to Cdc5p, the single Polo kinase in budding yeast that regulates mitotic progression and cytokinesis. Dbf4p mediates an interaction with the Polo substrate-binding domain to inhibit its essential role during mitosis. Although Dbf4p does not inhibit Polo kinase activity, it nonetheless inhibits Polo-mediated activation of the mitotic exit network (MEN, presumably by altering Polo substrate targeting. In addition, although dbf4 mutants defective for interaction with Polo transit S-phase normally, they aberrantly segregate chromosomes following nuclear misorientation. Therefore, Cdc7p-Dbf4p prevents inappropriate exit from mitosis by inhibiting Polo kinase and functions in the spindle position checkpoint.

  10. Selective anticancer activity of a hexapeptide with sequence homology to a non-kinase domain of Cyclin Dependent Kinase 4

    OpenAIRE

    Agarwala Usha; Blaydes Jeremy P; Maurer Richard I; Essex Jon W; Kilburn Jeremy D; Warenius Hilmar M; Seabra Laurence A

    2011-01-01

    Abstract Background Cyclin-dependent kinases 2, 4 and 6 (Cdk2, Cdk4, Cdk6) are closely structurally homologous proteins which are classically understood to control the transition from the G1 to the S-phases of the cell cycle by combining with their appropriate cyclin D or cyclin E partners to form kinase-active holoenzymes. Deregulation of Cdk4 is widespread in human cancer, CDK4 gene knockout is highly protective against chemical and oncogene-mediated epithelial carcinogenesis, despite the c...

  11. Selective anticancer activity of a hexapeptide with sequence homology to a non-kinase domain of Cyclin Dependent Kinase 4

    Directory of Open Access Journals (Sweden)

    Agarwala Usha

    2011-06-01

    Full Text Available Abstract Background Cyclin-dependent kinases 2, 4 and 6 (Cdk2, Cdk4, Cdk6 are closely structurally homologous proteins which are classically understood to control the transition from the G1 to the S-phases of the cell cycle by combining with their appropriate cyclin D or cyclin E partners to form kinase-active holoenzymes. Deregulation of Cdk4 is widespread in human cancer, CDK4 gene knockout is highly protective against chemical and oncogene-mediated epithelial carcinogenesis, despite the continued presence of CDK2 and CDK6; and overexpresssion of Cdk4 promotes skin carcinogenesis. Surprisingly, however, Cdk4 kinase inhibitors have not yet fulfilled their expectation as 'blockbuster' anticancer agents. Resistance to inhibition of Cdk4 kinase in some cases could potentially be due to a non-kinase activity, as recently reported with epidermal growth factor receptor. Results A search for a potential functional site of non-kinase activity present in Cdk4 but not Cdk2 or Cdk6 revealed a previously-unidentified loop on the outside of the C'-terminal non-kinase domain of Cdk4, containing a central amino-acid sequence, Pro-Arg-Gly-Pro-Arg-Pro (PRGPRP. An isolated hexapeptide with this sequence and its cyclic amphiphilic congeners are selectively lethal at high doses to a wide range of human cancer cell lines whilst sparing normal diploid keratinocytes and fibroblasts. Treated cancer cells do not exhibit the wide variability of dose response typically seen with other anticancer agents. Cancer cell killing by PRGPRP, in a cyclic amphiphilic cassette, requires cells to be in cycle but does not perturb cell cycle distribution and is accompanied by altered relative Cdk4/Cdk1 expression and selective decrease in ATP levels. Morphological features of apoptosis are absent and cancer cell death does not appear to involve autophagy. Conclusion These findings suggest a potential new paradigm for the development of broad-spectrum cancer specific therapeutics with

  12. Xingshentongqiao Decoction Mediates Proliferation, Apoptosis, Orexin-A Receptor and Orexin-B Receptor Messenger Ribonucleic Acid Expression and Represses Mitogen-activated Protein Kinase Signaling

    Institute of Scientific and Technical Information of China (English)

    Yuanli Dong; Mei Li; Shaojie Wang; Yuwei Dong; Hongxia Zhao; Zhong Dai

    2015-01-01

    Background:Hypocretin (HCRT) signaling plays an important role in the pathogenesis of narcolepsy and can be significantly influenced by Chinese herbal therapy.Our previous study showed that xingshentongqiao decoction (XSTQ) is clinically effective for the treatment of narcolepsy.To determine whether XSTQ improves narcolepsy by modulating HCRT signaling,we investigated its effects on SH-SY5Y cell proliferation,apoptosis,and HCRT receptor 1/2 (orexin receptor 1 [OXl R] and orexin receptor 2 [OX2R]) expression.The signaling pathways involved in these processes were also assessed.Methods:The effects of XSTQ on proliferation and apoptosis in SH-SY5Y cells were assessed using cell counting kit-8 and annexin V-fluorescein isothiocyanate assays.OX1R and OX2R expression was assessed by quantitative real-time polymerase chain reaction analysis.Western blotting for mitogen-activated protein kinase (MAPK) pathway activation was performed to further assess the signaling mechanism of XSTQ.Results:XSTQ reduced the proliferation and induced apoptosis of SH-SY5Y cells.This effect was accompanied by the upregulation of OX 1R and OX2R expression and the reduced phosphorylation of extracellular signal-regulated kinase (Erk) 1/2,p38 MAPK and c-Jun N-terminal kinase (JNK).Conclusions:XSTQ inhibits proliferation and induces apoptosis in SH-SY5Y cells.XSTQ also promotes OX1R and OX2R expression.These effects are associated with the repression of the Erkl/2,p38 MAPK,and JNK signaling pathways.These results define a molecular mechanism for XSTQ in regulating HCRT and MAPK activation,which may explain its ability to treat narcolepsy.

  13. Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

    KAUST Repository

    Irving, Helen R.

    2012-02-01

    Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.

  14. Propranolol Improves Impaired Hepatic Phosphatidylinositol 3-Kinase/Akt Signaling after Burn Injury

    OpenAIRE

    Brooks, Natasha C; Song, Juquan; Boehning, Darren; Kraft, Robert; Finnerty, Celeste C.; Herndon, David N; Jeschke, Marc G.

    2012-01-01

    Severe burn injury is associated with induction of the hepatic endoplasmic reticulum (ER) stress response. ER stress leads to activation of c-Jun N-terminal kinase (JNK), suppression of insulin receptor signaling via phosphorylation of insulin receptor substrate 1 and subsequent insulin resistance. Marked and sustained increases in catecholamines are prominent after a burn. Here, we show that administration of propranolol, a nonselective β1/2 adrenergic receptor antagonist, attenuates ER stre...

  15. Cloning and expression of the heterodimeric deoxyguanosine kinase/deoxyadenosine kinase of Lactobacillus acidophilus R-26.

    Science.gov (United States)

    Ma, G T; Hong, Y S; Ives, D H

    1995-03-24

    Two uniquely paired deoxynucleoside kinases, deoxycytidine kinase/deoxyadenosine kinase (dCK/dAK) and deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) are required, together with thymidine kinase (TK), for deoxynucleotide synthesis in Lactobacillus acidophilus R-26. Using polymerase chain reaction-generated probes based on N-terminal amino acid sequences, we have cloned tandem genes for 25- and 26-kDa polypeptides, whose derived amino acid sequences and size correspond to wild-type Lactobacillus enzyme subunits. Expression in Escherichia coli uses a single endogenous promoter and yields active dGK/dAK (approximately 3% of extracted protein) closely resembling wild-type dGK/dAK in specificity, kinetics, heterotropic activation, and end product inhibition. Alignment of cloned genes reveals 65% identity in their DNA sequences and 61% identity in derived amino acid sequences. Comparison with herpes-viral TKs reveals three conserved regions: glycine- and arginine-rich ATP-binding motifs and a D/E-R-S/H motif at the putative TK deoxynucleoside site. Greater homology, however, is seen upon multiple alignment of dGK with mammalian deoxycytidine kinases, yielding the consensus sequence-D/E-R-S-I/V-Y-x-D-.dGK also shares a sequence (-Y-D-P-T-I/L-E-D-S/Y-Y-) required for GTP hydrolysis by p21ras.

  16. Structural Bioinformatics and Protein Docking Analysis of the Molecular Chaperone-Kinase Interactions: Towards Allosteric Inhibition of Protein Kinases by Targeting the Hsp90-Cdc37 Chaperone Machinery

    Directory of Open Access Journals (Sweden)

    Gennady Verkhivker

    2013-11-01

    Full Text Available A fundamental role of the Hsp90-Cdc37 chaperone system in mediating maturation of protein kinase clients and supporting kinase functional activity is essential for the integrity and viability of signaling pathways involved in cell cycle control and organism development. Despite significant advances in understanding structure and function of molecular chaperones, the molecular mechanisms and guiding principles of kinase recruitment to the chaperone system are lacking quantitative characterization. Structural and thermodynamic characterization of Hsp90-Cdc37 binding with protein kinase clients by modern experimental techniques is highly challenging, owing to a transient nature of chaperone-mediated interactions. In this work, we used experimentally-guided protein docking to probe the allosteric nature of the Hsp90-Cdc37 binding with the cyclin-dependent kinase 4 (Cdk4 kinase clients. The results of docking simulations suggest that the kinase recognition and recruitment to the chaperone system may be primarily determined by Cdc37 targeting of the N-terminal kinase lobe. The interactions of Hsp90 with the C-terminal kinase lobe may provide additional “molecular brakes” that can lock (or unlock kinase from the system during client loading (release stages. The results of this study support a central role of the Cdc37 chaperone in recognition and recruitment of the kinase clients. Structural analysis may have useful implications in developing strategies for allosteric inhibition of protein kinases by targeting the Hsp90-Cdc37 chaperone machinery.

  17. Light induces Fos expression via extracellular signal-regulated kinases 1/2 in melanopsin-expressing PC12 cells

    DEFF Research Database (Denmark)

    Moldrup, Marie-Louise Bülow; Georg, Birgitte; Falktoft, Birgitte;

    2010-01-01

    -regulated protein kinase 1/2 (ERK1/2) was found as pharmacological blockage of this kinase suppressed the light-induced Fos expression. Illumination increased the inositol phosphate turnover and induced phosphorylation of ERK1/2 and p38 but not the c-Jun N-terminal kinase. The Galpha(q/11) protein inhibitor YM......254890 attenuated these intracellular light responses. Our data strongly indicate that Galpha(q/11)-mediated ERK1/2 activation is essential for expression of Fos upon illumination of melanopsin-expressing PC12 cells....

  18. Horse chestnut extract induces contraction force generation in fibroblasts through activation of Rho/Rho kinase.

    Science.gov (United States)

    Fujimura, Tsutomu; Moriwaki, Shigeru; Hotta, Mitsuyuki; Kitahara, Takashi; Takema, Yoshinori

    2006-06-01

    Contraction forces generated by non-muscle cells such as fibroblasts play important roles in determining cell morphology, vasoconstriction, and/or wound healing. However, few factors that induce cell contraction forces are known, such as lysophosphatidic acid and thrombin. Our study analyzed various plant extracts for ingredients that induce generation of cell contraction forces in fibroblasts populating collagen gels. We found that an extract of Horse chestnut (Aesculus hippocastanum) is able to induce such contraction forces in fibroblasts. The involvement of actin polymerization and stress fiber formation in the force generation was suggested by inhibition of this effect by cytochalasin D and by Rhodamine phalloidin. Rho kinase inhibitors (Y27632 and HA1077) and a Rho inhibitor (exoenzyme C3) significantly inhibited the force generation induced by the Horse chestnut extract. H7, which inhibits Rho kinase as well as other protein kinases, also significantly inhibited induction of force generation. However, inhibitors of other protein kinases such as myosin light chain kinase (ML-9), protein kinase C (Calphostin), protein kinase A (KT5720), and tyrosine kinase (Genistein, Herbimycin A) had no effect on force generation induced by Horse chestnut extract. These results suggest that the Horse chestnut extract induces generation of contraction forces in fibroblasts through stress fiber formation followed by activation of Rho protein and Rho kinase but not myosin light chain kinase or other protein kinases. PMID:16754996

  19. Mechanisms of Activation of Receptor Tyrosine Kinases: Monomers or Dimers

    Directory of Open Access Journals (Sweden)

    Ichiro N. Maruyama

    2014-04-01

    Full Text Available Receptor tyrosine kinases (RTKs play essential roles in cellular processes, including metabolism, cell-cycle control, survival, proliferation, motility and differentiation. RTKs are all synthesized as single-pass transmembrane proteins and bind polypeptide ligands, mainly growth factors. It has long been thought that all RTKs, except for the insulin receptor (IR family, are activated by ligand-induced dimerization of the receptors. An increasing number of diverse studies, however, indicate that RTKs, previously thought to exist as monomers, are present as pre-formed, yet inactive, dimers prior to ligand binding. The non-covalently associated dimeric structures are reminiscent of those of the IR family, which has a disulfide-linked dimeric structure. Furthermore, recent progress in structural studies has provided insight into the underpinnings of conformational changes during the activation of RTKs. In this review, I discuss two mutually exclusive models for the mechanisms of activation of the epidermal growth factor receptor, the neurotrophin receptor and IR families, based on these new insights.

  20. Ethnic differences in tissue creatine kinase activity: an observational study.

    Directory of Open Access Journals (Sweden)

    Lizzy M Brewster

    Full Text Available BACKGROUND: Serum creatine kinase (CK levels are reported to be around 70% higher in healthy black people, as compared to white people (median value 88 IU/L in white vs 149 IU/L in black people. As serum CK in healthy people is thought to occur from a proportional leak from normal tissues, we hypothesized that the black population subgroup has a generalized higher CK activity in tissues. METHODOLOGY/PRINCIPAL FINDINGS: We compared CK activity spectrophotometrically in tissues with high and fluctuating energy demands including cerebrum, cerebellum, heart, renal artery, and skeletal muscle, obtained post-mortem in black and white men. Based on serum values, we conservatively estimated to find a 50% greater CK activity in black people compared with white people, and calculated a need for 10 subjects of one gender in each group to detect this difference. We used mixed linear regression models to assess the possible influence of ethnicity on CK activity in different tissues, with ethnicity as a fixed categorical subject factor, and CK of different tissues clustered within one person as the repeated effect response variable. We collected post-mortem tissue samples from 17 white and 10 black males, mean age 62 y (SE 4. Mean tissue CK activity was 76% higher in tissues from black people (estimated marginal means 107.2 [95% CI, 76.7 to 137.7] mU/mg protein in white, versus 188.6 [148.8 to 228.4] in black people, p = 0.002. CONCLUSION: We found evidence that black people have higher CK activity in all tissues with high and fluctuating energy demands studied. This finding may help explain the higher serum CK levels found in this population subgroup. Furthermore, our data imply that there are differences in CK-dependent ATP buffer capacity in tissue between the black and the white population subgroup, which may become apparent with high energy demands.

  1. Berberine Promotes Glucose Consumption Independently of AMP-Activated Protein Kinase Activation

    OpenAIRE

    Miao Xu; Yuanyuan Xiao; Jun Yin; Wolin Hou; Xueying Yu; Li Shen; Fang Liu; Li Wei; Weiping Jia

    2014-01-01

    Berberine is a plant alkaloid with anti-diabetic action. Activation of AMP-activated protein kinase (AMPK) pathway has been proposed as mechanism for berberine's action. This study aimed to examine whether AMPK activation was necessary for berberine's glucose-lowering effect. We found that in HepG2 hepatocytes and C2C12 myotubes, berberine significantly increased glucose consumption and lactate release in a dose-dependent manner. AMPK and acetyl coenzyme A synthetase (ACC) phosphorylation wer...

  2. Involvement of Hypothalamic AMP-Activated Protein Kinase in Leptin-Induced Sympathetic Nerve Activation

    OpenAIRE

    Mamoru Tanida; Naoki Yamamoto; Toshishige Shibamoto; Kamal Rahmouni

    2013-01-01

    In mammals, leptin released from the white adipose tissue acts on the central nervous system to control feeding behavior, cardiovascular function, and energy metabolism. Central leptin activates sympathetic nerves that innervate the kidney, adipose tissue, and some abdominal organs in rats. AMP-activated protein kinase (AMPK) is essential in the intracellular signaling pathway involving the activation of leptin receptors (ObRb). We investigated the potential of AMPKα2 in the sympathetic effec...

  3. Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the γ1 subunit

    OpenAIRE

    Wu, Chongming; Guo, Yanshen; Su, Yan; Zhang, Xue; Luan, Hong; Zhang, Xiaopo; Zhu, Huixin; He, Huixia; Wang, Xiaoliang; Sun, Guibo; Sun, Xiaobo; Guo, Peng; Zhu, Ping

    2013-01-01

    Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP-activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin-induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)-elicited intracellular lipid accumulation and increased AMPK activity in...

  4. N-terminal cleavage and release of the ectodomain of Flt1 is mediated via ADAM10 and ADAM 17 and regulated by VEGFR2 and the Flt1 intracellular domain.

    Directory of Open Access Journals (Sweden)

    Nandita S Raikwar

    Full Text Available Flt is one of the cell surface VEGF receptors which can be cleaved to release an N-terminal extracellular fragment which, like alternately transcribed soluble Flt1 (sFlt1, can antagonize the effects of VEGF. In HUVEC and in HEK293 cells where Flt1 was expressed, metalloprotease inhibitors reduced Flt1 N-terminal cleavage. Overexpression of ADAM10 and ADAM17 increased cleavage while knockdown of ADAM10 and ADAM17 reduced N-terminal cleavage suggesting that these metalloproteases were responsible for Flt1 cleavage. Protein kinase C (PKC activation increased the abundance and the cleavage of Flt1 but this did not require any residues within the intracellular portion of Flt1. ALLN, a proteasomal inhibitor, increased the abundance of Flt1 which was additive to the effect of PKC. Removal of the entire cytosolic region of Flt1 appeared to stimulate cleavage of Flt1 and Flt1 was no longer sensitive to ALLN suggesting that the cytosolic region contained a degradation domain. Knock down of c-CBL, a ring finger ubiquitin ligase, in HEK293 cells increased the expression of Flt1 although it did not appear to require a previously published tyrosine residue (1333Y in the C-terminus of Flt1. Increasing VEGFR2 expression increased VEGF-stimulated sFlt1 expression and progressively reduced the cleavage of Flt1 with Flt1 staying bound to VEGFR2 as a heterodimer. Our results imply that secreted sFlt1 and cleaved Flt1 will tend to have local effects as a VEGF antagonist when released from cells expressing VEGFR2 and more distant effects when released from cells lacking VEGFR2.

  5. Exercise in rats does not alter hypothalamic AMP-activated protein kinase activity

    DEFF Research Database (Denmark)

    Andersson, Ulrika; Treebak, Jonas Thue; Nielsen, Jakob Nis;

    2005-01-01

    Recent studies have demonstrated that AMP-activated protein kinase (AMPK) in the hypothalamus is involved in the regulation of food intake. Because exercise is known to influence appetite and cause substrate depletion, it may also influence AMPK in the hypothalamus. Male rats that either rested...

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

    Energy Technology Data Exchange (ETDEWEB)

    Qiu, Wei; Wernimont, Amy; Tang, Keliang; Taylor, Sonya; Lunin, Vladimir; Schapira, Matthieu; Fentress, Sarah; Hui, Raymond; Sibley, L. David; (Toronto); (WU-MED)

    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. 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.

  7. Redox regulation of the AMP-activated protein kinase.

    Directory of Open Access Journals (Sweden)

    Yingying Han

    Full Text Available Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death.The aim of this study is to determine if AMP-activated protein kinase (AMPK, a cellular energy sensor, is activated by oxidants generated by Berberine in endothelial cells (EC.Bovine aortic endothelial cells (BAEC were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation.In BAEC, Berberine caused a dose- and time-dependent increase in the phosphorylation of AMPK at Thr172 and acetyl CoA carboxylase (ACC at Ser79, a well characterized downstream target of AMPK. Concomitantly, Berberine increased peroxynitrite, a potent oxidant formed by simultaneous generation of superoxide and nitric oxide. Pre-incubation of BAEC with anti-oxidants markedly attenuated Berberine-enhanced phosphorylation of both AMPK and ACC. Consistently, adenoviral expression of superoxide dismutase and pretreatment of L-N(G-Nitroarginine methyl ester (L-NAME; a non-selective NOS inhibitor blunted Berberine-induced phosphorylation of AMPK. Furthermore, mitochondria-targeted tempol (mito-tempol pretreatment or expression of uncoupling protein attenuated AMPK activation caused by Berberine. Depletion of mitochondria abolished the effects of Berberine on AMPK in EC. Finally, Berberine significantly increased the phosphorylation of LKB1 at Ser307 and gene silencing of LKB1 attenuated Berberine-enhanced AMPK Thr172 phosphorylation in BAEC.Our results suggest that mitochondria-derived superoxide anions and peroxynitrite are required for Berberine-induced AMPK activation in endothelial cells.

  8. Structures of down syndrome kinases, DYRKs, reveal mechanisms of kinase activation and substrate recognition

    DEFF Research Database (Denmark)

    Soundararajan, M.; Roos, A.K.; Savitsky, P.;

    2013-01-01

    Dual-specificity tyrosine-(Y)-phosphorylation-regulated kinases (DYRKs) play key roles in brain development, regulation of splicing, and apoptosis, and are potential drug targets for neurodegenerative diseases and cancer. We present crystal structures of one representative member of each DYRK sub...

  9. Role of the N-terminal seven residues of surfactant protein B (SP-B.

    Directory of Open Access Journals (Sweden)

    Mahzad Sharifahmadian

    Full Text Available Breathing is enabled by lung surfactant, a mixture of proteins and lipids that forms a surface-active layer and reduces surface tension at the air-water interface in lungs. Surfactant protein B (SP-B is an essential component of lung surfactant. In this study we probe the mechanism underlying the important functional contributions made by the N-terminal 7 residues of SP-B, a region sometimes called the "insertion sequence". These studies employed a construct of SP-B, SP-B (1-25,63-78, also called Super Mini-B, which is a 41-residue peptide with internal disulfide bonds comprising the N-terminal 7-residue insertion sequence and the N- and C-terminal helices of SP-B. Circular dichroism, solution NMR, and solid state (2H NMR were used to study the structure of SP-B (1-25,63-78 and its interactions with phospholipid bilayers. Comparison of results for SP-B (8-25,63-78 and SP-B (1-25,63-78 demonstrates that the presence of the 7-residue insertion sequence induces substantial disorder near the centre of the lipid bilayer, but without a major disruption of the overall mechanical orientation of the bilayers. This observation suggests the insertion sequence is unlikely to penetrate deeply into the bilayer. The 7-residue insertion sequence substantially increases the solution NMR linewidths, most likely due to an increase in global dynamics.

  10. Calmodulin binds to and inhibits the activity of phosphoglycerate kinase.

    Science.gov (United States)

    Myre, Michael A; O'Day, Danton H

    2004-09-17

    Phosphoglycerate kinase (PGK) functions as a cytoplasmic ATP-generating glycolytic enzyme, a nuclear mediator in DNA replication and repair, a stimulator of Sendai virus transcription and an extracellular disulfide reductase in angiogenesis. Probing of a developmental expression library from Dictyostelium discoideum with radiolabelled calmodulin led to the isolation of a cDNA encoding a putative calmodulin-binding protein (DdPGK) with 68% sequence similarity to human PGK. Dictyostelium, rabbit and yeast PGKs bound to calmodulin-agarose in a calcium-dependent manner while DdPGK constructs lacking the calmodulin-binding domain (209KPFLAILGGAKVSDKIKLIE228) failed to bind. The calmodulin-binding domain shows 80% identity between diverse organisms and is situated beside the hinge and within the ATP binding domain adjacent to nine mutations associated with PGK deficiency. Calmodulin addition inhibits yeast PGK activity in vitro while the calmodulin antagonist W-7 abrogates this inhibition. Together, these data suggest that PGK activity may be negatively regulated by calcium and calmodulin signalling in eukaryotic cells. PMID:15363631

  11. The role of AMP-activated protein kinase in regulation of skeletal muscle metabolism

    OpenAIRE

    Anna Dziewulska; Paweł Dobrzyń; Agnieszka Dobrzyń

    2010-01-01

    AMP-activated protein kinase (AMPK) is a conserved, ubiquitously expressed eukaryotic enzyme that is activated in response to increasing AMP level. Regulation of AMPK activity in skeletal muscle is coordinated by contraction and phosphorylation by upstream kinases and a growing number of hormones and cytokines. Once activated, AMPK turns on catabolic, ATP-generating pathways, and turns off ATP-consuming metabolic processes such as biosynthesis and proliferation. Activation of AMPK promotes gl...

  12. Pto kinase binds two domains of AvrPtoB and its proximity to the effector E3 ligase determines if it evades degradation and activates plant immunity.

    Directory of Open Access Journals (Sweden)

    Johannes Mathieu

    2014-07-01

    Full Text Available The tomato--Pseudomonas syringae pv. tomato (Pst--pathosystem is one of the best understood models for plant-pathogen interactions. Certain wild relatives of tomato express two closely related members of the same kinase family, Pto and Fen, which recognize the Pst virulence protein AvrPtoB and activate effector-triggered immunity (ETI. AvrPtoB, however, contains an E3 ubiquitin ligase domain in its carboxyl terminus which causes degradation of Fen and undermines its ability to activate ETI. In contrast, Pto evades AvrPtoB-mediated degradation and triggers ETI in response to the effector. It has been reported recently that Pto has higher kinase activity than Fen and that this difference allows Pto to inactivate the E3 ligase through phosphorylation of threonine-450 (T450 in AvrPtoB. Here we show that, in contrast to Fen which can only interact with a single domain proximal to the E3 ligase of AvrPtoB, Pto binds two distinct domains of the effector, the same site as Fen and another N-terminal domain. In the absence of E3 ligase activity Pto binds to either domain of AvrPtoB to activate ETI. However, the presence of an active E3 ligase domain causes ubiquitination of Pto that interacts with the domain proximal to the E3 ligase, identical to ubiquitination of Fen. Only when Pto binds its unique distal domain can it resist AvrPtoB-mediated degradation and activate ETI. We show that phosphorylation of T450 is not required for Pto-mediated resistance in vivo and that a kinase-inactive version of Pto is still capable of activating ETI in response to AvrPtoB. Our results demonstrate that the ability of Pto to interact with a second site distal to the E3 ligase domain in AvrPtoB, and not a higher kinase activity or T450 phosphorylation, allows Pto to evade ubiquitination and to confer immunity to Pst.

  13. Rho-kinase inhibition ameliorates metabolic disorders through activation of AMPK pathway in mice.

    Directory of Open Access Journals (Sweden)

    Kazuki Noda

    Full Text Available BACKGROUND: Metabolic disorders, caused by excessive calorie intake and low physical activity, are important cardiovascular risk factors. Rho-kinase, an effector protein of the small GTP-binding protein RhoA, is an important cardiovascular therapeutic target and its activity is increased in patients with metabolic syndrome. We aimed to examine whether Rho-kinase inhibition improves high-fat diet (HFD-induced metabolic disorders, and if so, to elucidate the involvement of AMP-activated kinase (AMPK, a key molecule of metabolic conditions. METHODS AND RESULTS: Mice were fed a high-fat diet, which induced metabolic phenotypes, such as obesity, hypercholesterolemia and glucose intolerance. These phenotypes are suppressed by treatment with selective Rho-kinase inhibitor, associated with increased whole body O2 consumption and AMPK activation in the skeletal muscle and liver. Moreover, Rho-kinase inhibition increased mRNA expression of the molecules linked to fatty acid oxidation, mitochondrial energy production and glucose metabolism, all of which are known as targets of AMPK in those tissues. In systemic overexpression of dominant-negative Rho-kinase mice, body weight, serum lipid levels and glucose metabolism were improved compared with littermate control mice. Furthermore, in AMPKα2-deficient mice, the beneficial effects of fasudil, a Rho-kinase inhibitor, on body weight, hypercholesterolemia, mRNA expression of the AMPK targets and increase of whole body O2 consumption were absent, whereas glucose metabolism was restored by fasudil to the level in wild-type mice. In cultured mouse myocytes, pharmacological and genetic inhibition of Rho-kinase increased AMPK activity through liver kinase b1 (LKB1, with up-regulation of its targets, which effects were abolished by an AMPK inhibitor, compound C. CONCLUSIONS: These results indicate that Rho-kinase inhibition ameliorates metabolic disorders through activation of the LKB1/AMPK pathway, suggesting that

  14. Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture

    DEFF Research Database (Denmark)

    Biondi, R M; Engel, M; Sauane, M;

    1996-01-01

    that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping-pong mechanism of catalysis: its autophosphorylation. Upon in vivo 32P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated......Although a number of nucleoside diphosphate kinases (NDPKs) have been reported to act as inhibitors of metastasis or as a transcription factor in mammals, it is not known whether these functions are linked to their enzymatic activity or how this protein is regulated. In this report, we show...... on histidine residues, however, only the B isoform appeared to be serine phosphorylated....

  15. P34^ Kinase and MAP Kinase Activities and Parthenogenetic Activation in Porcine Oocytes after Injection of Miniature Pig Sperm Extracts

    OpenAIRE

    Matsuura, Daizou; Maeda, Teruo

    2008-01-01

    The aim of the present study was to examine the rate of activation and time-dependent changes in p34cdc2 kinase and MAP kinase activities in porcine oocytes after injection of sperm extracts (SE) or treatment with Ca2+ ionophore to clarify whether SE injection is useful for porcine oocyte activation. SE was prepared from miniature pig sperm by non-ionic surfactant. Oocytes that were treated with Ca2+ ionophore and injected with SE were activated at rates of 41% and 46%, respectively. The acti...

  16. Reaction of the N-terminal methionine residues in cyanase with diethylpyrocarbonate.

    Science.gov (United States)

    Anderson, P M; Korte, J J; Holcomb, T A

    1994-11-29

    Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The enzyme is a decamer of identical subunits (M(r) = 17,000). Previous studies have shown that modification of either the single cysteine residue or the single histidine residue in each subunit gives an active decameric derivative that dissociates reversibly to inactive dimer derivative, indicating that decameric structure is required for activity and that the SH and imidazole groups are not required for catalytic activity [Anderson, P. M., Korte, J. J., Holcomb, T. A., Cho, Y.-G., Son, C.-M., & Sung, Y.-C. (1994) J. Biol. Chem. 269, 15036-15045]. Here the effects of reaction of the reagent diethylpyrocarbonate (DEPC) with cyanase or mutant cyanases are reported. DEPC reacts stoichiometrically with the histidine residue and at one additional site in each subunit when the enzyme is in the inactive dimer form, preventing reactivation. DEPC reacts stoichiometrically (with the same result on reactivation) at only one site per subunit with the inactive dimer form of cyanase mutants in which the single histidine residue has been replaced by one of several different amino acids by site-directed mutagenesis; the site of the reaction was identified as the amino group of the N-terminal methionine. DEPC does not react with the histidine residue of the active decameric form of wild-type cyanase and does not affect activity of the active decameric form of wild-type or mutant cyanases. Reaction with the N-terminal amino group of methionine apparently prevents reactivation of the mutant enzymes by blocking association to decamer.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. ELKS controls the pool of readily releasable vesicles at excitatory synapses through its N-terminal coiled-coil domains.

    Science.gov (United States)

    Held, Richard G; Liu, Changliang; Kaeser, Pascal S

    2016-06-02

    In a presynaptic nerve terminal, synaptic strength is determined by the pool of readily releasable vesicles (RRP) and the probability of release (P) of each RRP vesicle. These parameters are controlled at the active zone and vary across synapses, but how such synapse specific control is achieved is not understood. ELKS proteins are enriched at vertebrate active zones and enhance P at inhibitory hippocampal synapses, but ELKS functions at excitatory synapses are not known. Studying conditional knockout mice for ELKS, we find that ELKS enhances the RRP at excitatory synapses without affecting P. Surprisingly, ELKS C-terminal sequences, which interact with RIM, are dispensable for RRP enhancement. Instead, the N-terminal ELKS coiled-coil domains that bind to Liprin-α and Bassoon are necessary to control RRP. Thus, ELKS removal has differential, synapse-specific effects on RRP and P, and our findings establish important roles for ELKS N-terminal domains in synaptic vesicle priming.

  18. [Chemical synthesis of lactococcin B and functional evaluation of the N-terminal domain using a truncated synthetic analogue].

    Science.gov (United States)

    Lasta, S; Fajloun, Z; Mansuelle, P; Sabatier, J M; Boudabous, A; Sampieri, F

    2008-01-01

    The lactococcin B (LnB) is a hydrophobic, positively charged bacteriocin, produced by Lactococcus lactis ssp. cremoris 9B4. It consists of a peptidic chain made up of 47 amino acid residues, and inhibits Lactococcus exclusively. In order to study its biological activity a synthetic lactococcin B (LnBs) was obtained by solid-phase chemical synthesis using a Fmoc strategy. LnBs was shown to be indistinguishable from the natural peptide. In addition, a synthetic (7-47) LnBst analogue was obtained by withdrawal of peptidyl-resin after the 41 cycle of LnBs peptide chain assembly. The synthetic N-terminal truncated (7-47) LnBst analogue was found to be inactive on indicator strains. Our results strongly suggest that the first six N-terminal amino acid residues are involved in the bactericidal activity of LnB.

  19. Protein kinase A binds and activates heat shock factor 1.

    Directory of Open Access Journals (Sweden)

    Ayesha Murshid

    Full Text Available BACKGROUND: Many inducible transcription factors are regulated through batteries of posttranslational modifications that couple their activity to inducing stimuli. We have studied such regulation of Heat Shock Factor 1 (HSF1, a key protein in control of the heat shock response, and a participant in carcinogenisis, neurological health and aging. As the mechanisms involved in the intracellular regulation of HSF1 in good health and its dysregulation in disease are still incomplete we are investigating the role of posttranslational modifications in such regulation. METHODOLOGY/PRINCIPAL FINDINGS: In a proteomic study of HSF1 binding partners, we have discovered its association with the pleiotropic protein kinase A (PKA. HSF1 binds avidly to the catalytic subunit of PKA, (PKAcα and becomes phosphorylated on a novel serine phosphorylation site within its central regulatory domain (serine 320 or S320, both in vitro and in vivo. Intracellular PKAcα levels and phosphorylation of HSF1 at S320 were both required for HSF1 to be localized to the nucleus, bind to response elements in the promoter of an HSF1 target gene (hsp70.1 and activate hsp70.1 after stress. Reduction in PKAcα levels by small hairpin RNA led to HSF1 exclusion from the nucleus, its exodus from the hsp70.1 promoter and decreased hsp70.1 transcription. Likewise, null mutation of HSF1 at S320 by alanine substitution for serine led to an HSF1 species excluded from the nucleus and deficient in hsp70.1 activation. CONCLUSIONS: These findings of PKA regulation of HSF1 through S320 phosphorylation add to our knowledge of the signaling networks converging on this factor and may contribute to elucidating its complex roles in the stress response and understanding HSF1 dysregulation in disease.

  20. Hypoxia inhibits colonic ion transport via activation of AMP kinase.

    LENUS (Irish Health Repository)

    Collins, Danielle

    2012-02-01

    BACKGROUND AND AIMS: Mucosal hypoxia is a common endpoint for many pathological processes including ischemic colitis, colonic obstruction and anastomotic failure. Previous studies suggest that hypoxia modulates colonic mucosal function through inhibition of chloride secretion. However, the molecular mechanisms underlying this observation are poorly understood. AMP-activated protein kinase (AMPK) is a metabolic energy regulator found in a wide variety of cells and has been linked to cystic fibrosis transmembrane conductance regulator (CFTR) mediated chloride secretion in several different tissues. We hypothesized that AMPK mediates many of the acute effects of hypoxia on human and rat colonic electrolyte transport. METHODS: The fluorescent chloride indicator dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide was used to measure changes in intracellular chloride concentrations in isolated single rat colonic crypts. Ussing chamber experiments in human colonic mucosa were conducted to evaluate net epithelial ion transport. RESULTS: This study demonstrates that acute hypoxia inhibits electrogenic chloride secretion via AMPK mediated inhibition of CFTR. Pre-treatment of tissues with the AMPK inhibitor 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyyrazolo [1,5-a] pyrimidine (compound C) in part reversed the effects of acute hypoxia on chloride secretion. CONCLUSION: We therefore suggest that AMPK is a key component of the adaptive cellular response to mucosal hypoxia in the colon. Furthermore, AMPK may represent a potential therapeutic target in diseased states or in prevention of ischemic intestinal injury.

  1. N-terminal protein processing: A comparative proteogenomic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Bonissone, Stefano; Gupta, Nitin; Romine, Margaret F.; Bradshaw, Ralph A.; Pevzner, Pavel A.

    2013-01-01

    N-Terminal Methionine Excision (NME) is a universally conserved mechanism with the same specificity across all life forms that removes the first Methionine in proteins when the second residue is Gly, Ala, Ser, Cys, Thr, Pro, or Val. In spite of its necessity for proper cell functioning, the functional role of NME remains unclear. In 1988, Arfin and Bradshaw connected NME with the N-end protein degradation rule and postulated that the role of NME is to expose the stabilizing residues with the goal to resist protein degradation. While this explanation (that treats 7 stabilizing residues in the same manner) has become the de facto dogma of NME, comparative proteogenomics analysis of NME tells a different story. We suggest that the primary role of NME is to expose only two (rather than seven) amino acids Ala and Ser for post-translational modifications (e.g., acetylation) rather than to regulate protein degradation. We argue that, contrary to the existing view, NME is not crucially important for proteins with 5 other stabilizing residue at the 2nd positions that are merely bystanders (their function is not affected by NME) that become exposed to NME because their sizes are comparable or smaller than the size of Ala and Ser.

  2. Activated Cdc42-associated kinase Ack1 promotes prostate cancer progression via androgen receptor tyrosine phosphorylation

    OpenAIRE

    Mahajan, Nupam P.; Liu, Yuanbo; Majumder, Samarpan; Warren, Maria R.; Parker, Carol E.; Mohler, James L.; Earp, H. Shelton; Whang, Young E.

    2007-01-01

    Activation of the androgen receptor (AR) may play a role in androgen-independent progression of prostate cancer. Multiple mechanisms of AR activation, including stimulation by tyrosine kinases, have been postulated. We and others have recently shown involvement of activated Cdc42-associated tyrosine kinase Ack1 in advanced human prostate cancer. Here we provide the molecular basis for interplay between Ack1 and AR in prostate cancer cells. Activated Ack1 promoted androgen-independent growth o...

  3. Quinoxaline-Based Scaffolds Targeting Tyrosine Kinases and Their Potential Anticancer Activity.

    Science.gov (United States)

    El Newahie, Aliya M S; Ismail, Nasser S M; Abou El Ella, Dalal A; Abouzid, Khaled A M

    2016-05-01

    Quinoxaline derivatives, also called benzopyrazines, are an important class of heterocyclic compounds. Quinoxalines have drawn great attention due to their wide spectrum of biological activities. They are considered as an important basis for anticancer drugs due to their potential activity as protein kinase inhibitors. In this review, we focus on the chemistry of the quinoxaline derivatives, the strategies for their synthesis, their potential activities against various tyrosine kinases, and on the structure-activity relationship studies reported to date.

  4. N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication

    Institute of Scientific and Technical Information of China (English)

    Yan-Chang Lei; Dong-Liang Yang; Yong-Jun Tian; Hong-Hui Ding; Bao-Ju Wang; Yan Yang; You-Hua Hao; Xi-Ping Zhao; Meng-Ji Lu; Fei-Li Gong

    2006-01-01

    AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G(APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo.METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA.The expression of hepatitis B virus core antigen (HBcAg)in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively.RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells,and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly,the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls.CONCLUSION: Our findings provide probably the first

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

    Energy Technology Data Exchange (ETDEWEB)

    Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty; Lin, Yu-Hui; Amani, Mehrnaz; Allali-Hassani, Abdellah; Senisterra, Guillermo; Vedadi, Masoud; Tempel, Wolfram; Mackenzie, Farrell; Chau, Irene; Lourido, Sebastian; Sibley, L. David; Hui, Raymond (Toronto); (WU-MED)

    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 binding 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.

  6. Rapid mitogen-activated protein kinase by basic fibroblast growth factor in rat intestin after ischemia/reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Xiao-Bing Fu; Yin-Hui Yang; Tong-Zhu Sun; Wei Chen; Jun-You Li; Zhi-Yong Sheng

    2003-01-01

    AIM: Previous studies showed that exogenous basic fibroblast growth factor (bFGF or FGF-2) could improve physiological dysfunction after intestinal ischemia/reperfusion (I/R) injury. However, the mechanisms of this protective effect of bFGF are still unclear. The present study was to detect the effect of bFGF on the activities of mitogen-activated protein kinase (MlAPK) signaling pathway in rat intestine after I/R injury, and to investigate the protective mechanisms of bFGF on intestinal ischemia injury. METttODS: Rat intestinal I/R injury was produced by clamping the superior mesenteric artery (SMA) for 45minutes and followed by repeffusion for 48 hours. Seventyeight Wistar rats were used and divided randomly into sham-operated group (A), normal saline control group (B),bFGF antibody pre-treated group (C), and bFGF treated group (D). Tn group A, SMA was separated without occlusion. In groups B, C and D, SMA was separated and occluded for 45 minutes, then, released for reperfusion for 48 hours. After the animals were sacrificed, blood and tissue samples were taken from the intestine 45 minutes after ischemia in group A and 2, 6, 24, and 48 hours after reperfusion in the other groups. Phosphorylated forms of p42/p44 MAPK, p38 MAPK and stress activated protein kinase/C-Jun N-terminal kinase (SAPK/JNK) were measured by immunohistochemistry. Plasma levels of D-lactate were examined and histological changes were observed under the light microscope. RESULTS: Intestinal I/R injury induced the expression of p42/p44 MAPK, p38 MAPK, and SAPK/JNK pathways and exogenous bFGF stimulated the early activation of p42/p44 MAPK and p38 MlAPK pathways. The expression of phosphorylated forms of p42/p44 MAPK was primarily localized in the nuclei of crypt cells and in the cytoplasm and nuclei of villus cells. The positive expression of p38MAPK was localized mainly in the nuclei of crypt cells, very few in villus cells. The activities of p42/p44 MAPK and p38MAPK peaked 6 hours after

  7. The MEKK1-MKK1/MKK2-MPK4 Kinase Cascade Negatively Regulates Immunity Mediated by a Mitogen-Activated Protein Kinase Kinase Kinase in Arabidopsis[C][W

    Science.gov (United States)

    Kong, Qing; Qu, Na; Gao, Minghui; Zhang, Zhibin; Ding, Xiaojun; Yang, Fan; Li, Yingzhong; Dong, Oliver X.; Chen, She; Li, Xin; Zhang, Yuelin

    2012-01-01

    In Arabidopsis thaliana, the MEKK1-MKK1/MKK2-MPK4 mitogen-activated protein (MAP) kinase cascade represses cell death and immune responses. In mekk1, mkk1 mkk2, and mpk4 mutants, programmed cell death and defense responses are constitutively activated, but the mechanism by which MEKK1, MKK1/MKK2, and MPK4 negatively regulate cell death and immunity was unknown. From a screen for suppressors of mkk1 mkk2, we found that mutations in suppressor of mkk1 mkk2 1 (summ1) suppress the cell death and defense responses not only in mkk1 mkk2 but also in mekk1 and mpk4. SUMM1 encodes the MAP kinase kinase kinase MEKK2. It interacts with MPK4 and is phosphorylated by MPK4 in vitro. Overexpression of SUMM1 activates cell death and defense responses that are dependent on the nucleotide binding–leucine-rich repeat protein SUMM2. Taken together, our data suggest that the MEKK1-MKK1/MKK2-MPK4 kinase cascade negatively regulates MEKK2 and activation of MEKK2 triggers SUMM2-mediated immune responses. PMID:22643122

  8. Structure of the N-terminal domain of the metalloprotease PrtV from Vibrio cholerae.

    Science.gov (United States)

    Edwin, Aaron; Persson, Cecilia; Mayzel, Maxim; Wai, Sun Nyunt; Öhman, Anders; Karlsson, B Göran; Sauer-Eriksson, A Elisabeth

    2015-12-01

    The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-protein that undergoes several N- and C-terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N-terminal domain (residues 23-103) that contains two short α-helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C-terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.

  9. Tor forms a dimer through an N-terminal helical solenoid with a complex topology.

    Science.gov (United States)

    Baretić, Domagoj; Berndt, Alex; Ohashi, Yohei; Johnson, Christopher M; Williams, Roger L

    2016-04-13

    The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor-Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor-Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor-Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended 'railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit.

  10. Tor forms a dimer through an N-terminal helical solenoid with a complex topology

    Science.gov (United States)

    Baretić, Domagoj; Berndt, Alex; Ohashi, Yohei; Johnson, Christopher M.; Williams, Roger L.

    2016-04-01

    The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor-Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor-Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor-Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended `railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit.

  11. Tor forms a dimer through an N-terminal helical solenoid with a complex topology

    Science.gov (United States)

    Baretić, Domagoj; Berndt, Alex; Ohashi, Yohei; Johnson, Christopher M.; Williams, Roger L.

    2016-01-01

    The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor–Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor–Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor–Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended ‘railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit. PMID:27072897

  12. Tor forms a dimer through an N-terminal helical solenoid with a complex topology.

    Science.gov (United States)

    Baretić, Domagoj; Berndt, Alex; Ohashi, Yohei; Johnson, Christopher M; Williams, Roger L

    2016-01-01

    The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor-Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor-Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor-Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended 'railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit. PMID:27072897

  13. Skeletal muscle Ca(2+)-independent kinase activity increases during either hypertrophy or running

    Science.gov (United States)

    Fluck, M.; Waxham, M. N.; Hamilton, M. T.; Booth, F. W.

    2000-01-01

    Spikes in free Ca(2+) initiate contractions in skeletal muscle cells, but whether and how they might signal to transcription factors in skeletal muscles of living animals is unknown. Since previous studies in non-muscle cells have shown that serum response factor (SRF) protein, a transcription factor, is phosphorylated rapidly by Ca(2+)/calmodulin (CaM)-dependent protein kinase after rises in intracellular Ca(2+), we measured enzymatic activity that phosphorylates SRF (designated SRF kinase activity). Homogenates from 7-day-hypertrophied anterior latissimus dorsi muscles of roosters had more Ca(2+)-independent SRF kinase activity than their respective control muscles. However, no differences were noted in Ca(2+)/CaM-dependent SRF kinase activity between control and trained muscles. To determine whether the Ca(2+)-independent and Ca(2+)/CaM-dependent forms of Ca(2+)/CaM-dependent protein kinase II (CaMKII) might contribute to some of the SRF kinase activity, autocamtide-3, a synthetic substrate that is specific for CaMKII, was employed. While the Ca(2+)-independent form of CaMKII was increased, like the Ca(2+)-independent form of SRF kinase, no alteration in CaMKII occurred at 7 days of stretch overload. These observations suggest that some of SRF phosphorylation by skeletal muscle extracts could be due to CaMKII. To determine whether this adaptation was specific to the exercise type (i.e., hypertrophy), similar measurements were made in the white vastus lateralis muscle of rats that had completed 2 wk of voluntary running. Although Ca(2+)-independent SRF kinase was increased, no alteration occurred in Ca(2+)/CaM-dependent SRF kinase activity. Thus any role of Ca(2+)-independent SRF kinase signaling has downstream modulators specific to the exercise phenotype.

  14. Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases.

    Directory of Open Access Journals (Sweden)

    Nikita Chopra

    2016-03-01

    Full Text Available Bruton's tyrosine kinase (Btk is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA. Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site.

  15. FgPrp4 Kinase Is Important for Spliceosome B-Complex Activation and Splicing Efficiency in Fusarium graminearum.

    Science.gov (United States)

    Gao, Xuli; Jin, Qiaojun; Jiang, Cong; Li, Yang; Li, Chaohui; Liu, Huiquan; Kang, Zhensheng; Xu, Jin-Rong

    2016-04-01

    PRP4 encodes the only kinase among the spliceosome components. Although it is an essential gene in the fission yeast and other eukaryotic organisms, the Fgprp4 mutant was viable in the wheat scab fungus Fusarium graminearum. Deletion of FgPRP4 did not block intron splicing but affected intron splicing efficiency in over 60% of the F. graminearum genes. The Fgprp4 mutant had severe growth defects and produced spontaneous suppressors that were recovered in growth rate. Suppressor mutations were identified in the PRP6, PRP31, BRR2, and PRP8 orthologs in nine suppressor strains by sequencing analysis with candidate tri-snRNP component genes. The Q86K mutation in FgMSL1 was identified by whole genome sequencing in suppressor mutant S3. Whereas two of the suppressor mutations in FgBrr2 and FgPrp8 were similar to those characterized in their orthologs in yeasts, suppressor mutations in Prp6 and Prp31 orthologs or FgMSL1 have not been reported. Interestingly, four and two suppressor mutations identified in FgPrp6 and FgPrp31, respectively, all are near the conserved Prp4-phosphorylation sites, suggesting that these mutations may have similar effects with phosphorylation by Prp4 kinase. In FgPrp31, the non-sense mutation at R464 resulted in the truncation of the C-terminal 130 aa region that contains all the conserved Prp4-phosphorylation sites. Deletion analysis showed that the N-terminal 310-aa rich in SR residues plays a critical role in the localization and functions of FgPrp4. We also conducted phosphoproteomics analysis with FgPrp4 and identified S289 as the phosphorylation site that is essential for its functions. These results indicated that FgPrp4 is critical for splicing efficiency but not essential for intron splicing, and FgPrp4 may regulate pre-mRNA splicing by phosphorylation of other components of the tri-snRNP although itself may be activated by phosphorylation at S289. PMID:27058959

  16. TYK2 kinase activity is required for functional type I interferon responses in vivo.

    Directory of Open Access Journals (Sweden)

    Michaela Prchal-Murphy

    Full Text Available Tyrosine kinase 2 (TYK2 is a member of the Janus kinase (JAK family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2(K923E mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein's stability. An inhibitory function was only observed upon over-expression of TYK2(K923Ein vitro. Tyk2(K923E mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors.

  17. Cln3-associated kinase activity in Saccharomyces cerevisiae is regulated by the mating factor pathway.

    Science.gov (United States)

    Jeoung, D I; Oehlen, L J; Cross, F R

    1998-01-01

    The Saccharomyces cerevisiae cell cycle is arrested in G1 phase by the mating factor pathway. Genetic evidence has suggested that the G1 cyclins Cln1, Cln2, and Cln3 are targets of this pathway whose inhibition results in G1 arrest. Inhibition of Cln1- and Cln2-associated kinase activity by the mating factor pathway acting through Far1 has been described. Here we report that Cln3-associated kinase activity is inhibited by mating factor treatment, with dose response and timing consistent with involvement in cell cycle arrest. No regulation of Cln3-associated kinase was observed in a fus3 kss1 strain deficient in mating factor pathway mitogen-activated protein (MAP) kinases. Inhibition occurs mainly at the level of specific activity of Cln3-Cdc28 complexes. Inhibition of the C-terminally truncated Cln3-1-associated kinase is not observed; such truncations were previously identified genetically as causing resistance to mating factor-induced cell cycle arrest. Regulation of Cln3-associated kinase specific activity by mating factor treatment requires Far1. Overexpression of Far1 restores inhibition of C-terminally truncated Cln3-1-associated kinase activity. G2/M-arrested cells are unable to regulate Cln3-associated kinase, possibly because of cell cycle regulation of Far1 abundance. Inhibition of Cln3-associated kinase activity by the mating factor pathway may allow this pathway to block the earliest step in normal cell cycle initiation, since Cln3 functions as the most upstream G1-acting cyclin, activating transcription of the G1 cyclins CLN1 and CLN2 as well as of the S-phase cyclins CLB5 and CLB6. PMID:9418890

  18. Induction of glutathione synthesis in human hepatocytes by acute and chronic arsenic exposure: Differential roles of mitogen-activated protein kinases

    International Nuclear Information System (INIS)

    Highlights: • Arsenic exposure increased intracellular levels of glutathione. • Mitogen-activated protein kinases were involved in glutathione homeostasis. • ERK contributed to glutathione synthesis during acute arsenic exposure. • Glutathione synthesis was regulated by p38 at least in part independent of NRF2 during chronic arsenic exposure. - Abstract: Glutathione (GSH) is a vital component of antioxidant defense which protects cells from toxic insults. Previously we found intracellular GSH was involved in cell resistance against arsenic-induced cytotoxicity. However, molecular mechanisms of GSH homeostasis during arsenic exposure are largely undefined. Here, we investigated roles of mitogen-activated protein kinases (MAPKs) in GSH synthesis pathway with two arsenic exposure strategies by using Chang human hepatocytes. In one strategy, acute arsenic exposure (20 μM, 24 h) was applied, as MAPK signaling is generally considered to be transient. In the other one, chronic arsenic exposure (500 nM, 20 weeks) was applied, which mimicked the general human exposure to arsenic. We found that acute arsenic exposure activated extracellular signal-regulated 1/2 kinases (ERK1/2) and c-Jun N-terminal kinase (JNK) in parallel with increased transcription and nuclear translocation of factor-erythroid 2-related factor 2 (NRF2) and enhanced expression of γ-glutamyl cysteine ligase catalytic subunit (GCLC), resulting in elevated intracellular GSH levels. Specific ERK inhibitor abolished arsenic-induced NRF2 nuclear translocation and GSH synthesis. During chronic arsenic exposure which induced a malignant cellular phenotype, continuous p38 activation and NRF2 nuclear translocation were observed with enhanced GSH synthesis. Specific p38 inhibitor attenuated arsenic-enhanced GSH synthesis without changing NRF2 nuclear translocation. Taken together, our results indicate MAPK pathways play an important role in cellular GSH homeostasis in response to arsenic. However, the

  19. Runx2 Trans-Activation Mediated by the Msx2-Interacting Nuclear Target Requires Homeodomain Interacting Protein Kinase-3

    OpenAIRE

    Sierra, Oscar L.; Towler, Dwight A.

    2010-01-01

    Runt-related transcription factor 2 (Runx2) and muscle segment homeobox homolog 2-interacting nuclear target (MINT) (Spen homolog) are transcriptional regulators critical for mammalian development. MINT enhances Runx2 activation of osteocalcin (OC) fibroblast growth factor (FGF) response element in an FGF2-dependent fashion in C3H10T1/2 cells. Although the MINT N-terminal RNA recognition motif domain contributes, the muscle segment homeobox homolog 2-interacting domain is sufficient for Runx2...

  20. Antral content, secretion and peripheral metabolism of N-terminal progastrin fragments

    DEFF Research Database (Denmark)

    Goetze, Jens Peter; Hansen, Carsten Palnaes; Rehfeld, Jens F

    2006-01-01

    OBJECTIVES: In addition to the acid-stimulatory gastrins, progastrin also release N-terminal fragments. In order to examine the cellular content, secretion and peripheral metabolism of these fragments, we developed an immunoassay specific for the N-terminal sequence of human progastrin. RESULTS: ...

  1. Enterovirus 71 VP1 activates calmodulin-dependent protein kinase II and results in the rearrangement of vimentin in human astrocyte cells.

    Directory of Open Access Journals (Sweden)

    Cong Haolong

    Full Text Available Enterovirus 71 (EV71 is one of the main causative agents of foot, hand and mouth disease. Its infection usually causes severe central nervous system diseases and complications in infected infants and young children. In the present study, we demonstrated that EV71 infection caused the rearrangement of vimentin in human astrocytoma cells. The rearranged vimentin, together with various EV71 components, formed aggresomes-like structures in the perinuclear region. Electron microscopy and viral RNA labeling indicated that the aggresomes were virus replication sites since most of the EV71 particles and the newly synthesized viral RNA were concentrated here. Further analysis revealed that the vimentin in the virus factories was serine-82 phosphorylated. More importantly, EV71 VP1 protein is responsible for the activation of calmodulin-dependent protein kinase II (CaMK-II which phosphorylated the N-terminal domain of vimentin on serine 82. Phosphorylation of vimentin and the formation of aggresomes were required for the replication of EV71 since the latter was decreased markedly after phosphorylation was blocked by KN93, a CaMK-II inhibitor. Thus, as one of the consequences of CaMK-II activation, vimentin phosphorylation and rearrangement may support virus replication by playing a structural role for the formation of the replication factories. Collectively, this study identified the replication centers of EV71 in human astrocyte cells. This may help us understand the replication mechanism and pathogenesis of EV71 in human.

  2. Enhanced biocontrol activity of Trichoderma through inactivation of a mitogen-activated protein kinase

    OpenAIRE

    Mendoza-Mendoza, Artemio; Pozo, María J.; Grzegorski, Darlene; Martínez, Pedro; García, Juan M.; Olmedo-Monfil, Vianey; Cortés, Carlos; Kenerley, Charles; Herrera-Estrella, Alfredo

    2003-01-01

    The production of lytic enzymes in Trichoderma is considered determinant in its parasitic response against fungal species. A mitogen-activated protein kinase encoding gene, tvk1, from Trichoderma virens was cloned, and its role during the mycoparasitism, conidiation, and biocontrol was examined in tvk1 null mutants. These mutants showed a clear increase in the level of the expression of mycoparasitism-related genes under simulated mycoparasitism and during direct confrontation with the plant ...

  3. Activation of mitogen-activated protein kinase pathway by extremely low-dose ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Keiji; Kodama, Seiji; Watanabe, Masami [Nagasaki Univ., Graduate School of Biomedical Sciences, Nagasaki (Japan)

    2003-07-01

    We demonstrated here that X-ray irradiation at very low doses of between 2 and 5 cGy stimulated activity of a member of mitogen-activated protein (MAP) kinase, the extracellular signal-regulated kinase (ERK) 1/2, in normal human diploid cells. Higher doses of irradiation at more than 1 Gy induced phosphorylation of ERK1/2 and accumulated p53 protein. Phosphorylation of ERK1/2 decreased with dose down to 50 cGy, however, doses of between 5 cGy and 2 cGy phosphorylated ERK1/2 as efficiently as higher doses of X-rays, while the p53 protein level was no longer changed by doses below 50 cGy. ATM-dependent phosphorylation of p53 protein at Ser15 and histone H2AX at Ser139 was only observed at higher doses at more than 10 cGy of X-rays. We found that MEK1 was phosphorylated with both 2 cGy and 6 Gy of X-rays, and that the MEK1 inhibitor, PD98059 decreased phosphorylation of the ERK1/2 proteins induced by 2 cGy or 6 Gy of X-rays. Similar suppressive effect was observed with the specific epidermal growth factor (EGF) receptor tyrosine kinase inhibitor, AG1478. These results indicate that a limited range of low dose ionizing radiation differentially activate ERK1/2 kinases via activation of EGF receptor and MEK, which mediates various effects of cells receiving very low doses of ionizing radiation. (author)

  4. INHIBITION OF IL-6-INDUCED STAT3 ACTIVATION IN MYELOMA CELLS BY PROTEIN KINASE A

    Institute of Scientific and Technical Information of China (English)

    宋伦; 黎燕; 沈倍奋

    2001-01-01

    To investigate the regulation effect of protein kinase A on IL-6-induced STAT3 activation in myeloma cells. Methods: Two human myeloma cell lines-Sko-007 and U266 were pretreated with Forskolin, a protein kinase A antagonist, and then stimulated by IL-6. The activation state of STAT3 in these two cells were examined by electrophoretic mobility shift assay (EMSA). Results: Although PKA pathway itself doesn't participate in IL-6 signal transduction in Sko-007 and U266 cells, activation of protein kinase A can inhibit IL-6-induced STAT3 activation in these two cell lines. Conclusion: There exists an inhibitory effect of protein kinase A on STAT3 activation in human myeloma cells treated by IL-6.

  5. The vasorelaxant effect of adrenomedullin, proadrenomedullin N-terminal 20 peptide and amylin in human skin

    DEFF Research Database (Denmark)

    Hasbak, Philip; Eskesen, Karen; Lind, Peter Henrik;

    2006-01-01

    of the peptides. The mRNA expression was assessed by real-time reverse transcriptase-polymerase chain reaction (real-time PCR). CGRP, adrenomedullin and amylin induced concentration-dependent, long-lasting increases in skin blood flow. The response to PAMP was shorter in duration appearing similar......In this study we aimed to assess in vivo, the vasodilator effects of adrenomedullin, proadrenomedullin N-terminal 20 peptide (PAMP) and amylin in human skin vasculature and compare the responses to the effects mediated by the endogenous neuropeptides calcitonin gene-related peptide (CGRP......) and substance P and to examine the mRNA expression of calcitonin receptor-like receptor (CL-R) and receptor-activity modifying proteins, RAMP1, RAMP 2 and RAMP3 in human subcutaneous arteries. Changes in skin blood flow of the forearm were measured using a Laser Doppler Imager after intradermal injection...

  6. Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

    Directory of Open Access Journals (Sweden)

    Ohannessian Arthur

    2004-05-01

    Full Text Available Abstract Background Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK. Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK. Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC lines. Methods Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. Results In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. Conclusions We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway.

  7. AMP-activated protein kinase (AMPK) activation regulates in vitro bone formation and bone mass

    OpenAIRE

    Shah, M; Kola, B; Bataveljic, A.; Arnett, T. R.; Viollet, B.; Saxon, L.; Korbonits, M.; C. Chenu

    2010-01-01

    Adenosine 5′-monophosphate-activated protein kinase (AMPK), a regulator of energy homeostasis, has a central role in mediating the appetite-modulating and metabolic effects of many hormones and antidiabetic drugs metformin and glitazones. The objective of this study was to determine if AMPK can be activated in osteoblasts by known AMPK modulators and if AMPK activity is involved in osteoblast function in vitro and regulation of bone mass in vivo. ROS 17/2.8 rat osteoblast-like cells were cult...

  8. Emerging Roles of AMP-Activated Protein Kinase

    DEFF Research Database (Denmark)

    Fritzen, Andreas Mæchel

    or has focused on specific physiological situations and tissues. The present PhD thesis has addressed the role of AMPK in regulation of: 1) substrate utilisation during and in recovery from exercise, 2) adipose tissue metabolism during weight loss, and 3) autophagy in skeletal muscle during exercise...... is an upstream kinase phosphorylating Unc51 like kinase 1 (ULK1) at Ser555, but this interaction per se seems not to be sufficient to change the autophagosome content. It cannot be excluded whether the AMPK-ULK1 association is important and necessary for regulation of autophagy and autophagosome biogenesis...

  9. The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease

    Science.gov (United States)

    Kereïche, Sami; Kováčik, Lubomír; Bednár, Jan; Pevala, Vladimír; Kunová, Nina; Ondrovičová, Gabriela; Bauer, Jacob; Ambro, Ľuboš; Bellová, Jana; Kutejová, Eva; Raška, Ivan

    2016-01-01

    Lon is an essential, multitasking AAA+ protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon’s N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning. PMID:27632940

  10. The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease.

    Science.gov (United States)

    Kereïche, Sami; Kováčik, Lubomír; Bednár, Jan; Pevala, Vladimír; Kunová, Nina; Ondrovičová, Gabriela; Bauer, Jacob; Ambro, Ľuboš; Bellová, Jana; Kutejová, Eva; Raška, Ivan

    2016-01-01

    Lon is an essential, multitasking AAA(+) protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon's N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning. PMID:27632940

  11. [Construction of the flavinogenic yeast Candida famata strains with high riboflavin kinase activity using gene engineering].

    Science.gov (United States)

    Ishchuk, O P; Iatsyshyn, V Iu; Dmytruk, K V; Voronovs'kyĭ, A Ia; Fedorovych, D V; Sybirnyĭ, A A

    2006-01-01

    The recombinant strains of the flavinogenic yeast Candida famata, which contain the DNA fragment consisting of the FMN1 gene (encoding the riboflavin kinase, enzyme that converts riboflavin to flavinmononucleotide) driven by the strong promoters (the regulated RIB1 or constitutive TEF1 promoter) were isolated. Riboflavin kinase activity in the isolated transformants was tested. The 6-8-fold increase of the riboflavin kinase activity was shown in the recombinant strains containing the integrated Debaryomyces hansenii FMN1 gene under the strong constitutive TEF1 promoter. The recombinant strains can be used for the following construction of flavinmononucleotide overproducers. PMID:17290783

  12. Aspirin Augments IgE-Mediated Histamine Release from Human Peripheral Basophils via Syk Kinase Activation

    Directory of Open Access Journals (Sweden)

    Hiroaki Matsuo

    2013-01-01

    Conclusions: Aspirin enhanced histamine release from basophils via increased Syk kinase activation, and that the augmentation of histamine release by NSAIDs or FAs may be one possible cause of worsening symptoms in patients with chronic urticaria and FDEIA.

  13. Structural Basis for Autoinhibition of c-Abl Tyrosine Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Nagar, Bhushan; Hantschel, Oliver; Young, Matthew A.; Scheffzek,Klaus; Veach, Darren; Bornmann, William; Clarkson, Bayard; Superti-Furga,Giulio; Kuriyan, John

    2003-03-21

    c-Abl is normally regulated by an autoinhibitory mechanism, the disruption of which leads to chronic myelogenous leukemia. The details of this mechanism have been elusive because c-Abl lacks aphosphotyrosine residue that triggers the assembly of the autoinhibited form of the closely related Src kinases by internally engaging the SH2 domain. Crystal structures of c-Abl show that the N-terminal myristoyl modification of c-Abl 1b binds to the kinase domain and induces conformational changes that allow the SH2 and SH3 domains to dock onto it. Autoinhibited c-Abl forms an assembly that is strikingly similar to that of inactive Src kinases but with specific differences that explain the differential ability of the drug STI-571/Gleevec/imatinib (STI-571)to inhibit the catalytic activity of Abl, but not that of c-Src.

  14. Kinase Activity Profiling of Gram-Negative Pneumonia

    NARCIS (Netherlands)

    Hoogendijk, Arie J.; Diks, Sander H.; Peppelenbosch, Maikel P.; van der Poll, Tom; Wieland, Catharina W.

    2011-01-01

    Pneumonia is a severe disease with high morbidity and mortality. A major causative pathogen is the Gram-negative bacterium Klebsiella (K.) pneumoniae Kinases play an integral role in the transduction of intracellular signaling cascades and regulate a diverse array of biological processes essential t

  15. Kinase activity profiling of gram-negative pneumonia

    NARCIS (Netherlands)

    A.J. Hoogendijk (Arie); S.H. Diks (Sander); M.P. Peppelenbosch (Maikel); T. van der Poll (Tom); C.W. Wieland (Catharina )

    2011-01-01

    textabstractPneumonia is a severe disease with high morbidity and mortality. A major causative pathogen is the Gram-negative bacterium Klebsiella (K.) pneumoniae. Kinases play an integral role in the transduction of intracellular signaling cascades and regulate a diverse array of biological processe

  16. Kinase activity profiling of gram-negative pneumonia

    NARCIS (Netherlands)

    A.J. Hoogendijk; S.H. Diks; M.P. Peppelenbosch; T. van der Poll; C.W. Wieland

    2011-01-01

    Pneumonia is a severe disease with high morbidity and mortality. A major causative pathogen is the Gram-negative bacterium Klebsiella (K.) pneumoniae. Kinases play an integral role in the transduction of intracellular signaling cascades and regulate a diverse array of biological processes essential

  17. Protective epitopes of the Plasmodium falciparum SERA5 malaria vaccine reside in intrinsically unstructured N-terminal repetitive sequences.

    Directory of Open Access Journals (Sweden)

    Masanori Yagi

    Full Text Available The malaria vaccine candidate antigen, SE36, is based on the N-terminal 47 kDa domain of Plasmodium falciparum serine repeat antigen 5 (SERA5. In epidemiological studies, we have previously shown the inhibitory effects of SE36 specific antibodies on in vitro parasite growth and the negative correlation between antibody level and malaria symptoms. A phase 1 b trial of the BK-SE36 vaccine in Uganda elicited 72% protective efficacy against symptomatic malaria in children aged 6-20 years during the follow-up period 130-365 days post-second vaccination. Here, we performed epitope mapping with synthetic peptides covering the whole sequence of SE36 to identify and map dominant epitopes in Ugandan adult serum presumed to have clinical immunity to P. falciparum malaria. High titer sera from the Ugandan adults predominantly reacted with peptides corresponding to two successive N-terminal regions of SERA5 containing octamer repeats and serine rich sequences, regions of SERA5 that were previously reported to have limited polymorphism. Affinity purified antibodies specifically recognizing the octamer repeats and serine rich sequences exhibited a high antibody-dependent cellular inhibition (ADCI activity that inhibited parasite growth. Furthermore, protein structure predictions and structural analysis of SE36 using spectroscopic methods indicated that N-terminal regions possessing inhibitory epitopes are intrinsically unstructured. Collectively, these results suggest that strict tertiary structure of SE36 epitopes is not required to elicit protective antibodies in naturally immune Ugandan adults.

  18. Protective epitopes of the Plasmodium falciparum SERA5 malaria vaccine reside in intrinsically unstructured N-terminal repetitive sequences.

    Science.gov (United States)

    Yagi, Masanori; Bang, Gilles; Tougan, Takahiro; Palacpac, Nirianne M Q; Arisue, Nobuko; Aoshi, Taiki; Matsumoto, Yoshitsugu; Ishii, Ken J; Egwang, Thomas G; Druilhe, Pierre; Horii, Toshihiro

    2014-01-01

    The malaria vaccine candidate antigen, SE36, is based on the N-terminal 47 kDa domain of Plasmodium falciparum serine repeat antigen 5 (SERA5). In epidemiological studies, we have previously shown the inhibitory effects of SE36 specific antibodies on in vitro parasite growth and the negative correlation between antibody level and malaria symptoms. A phase 1 b trial of the BK-SE36 vaccine in Uganda elicited 72% protective efficacy against symptomatic malaria in children aged 6-20 years during the follow-up period 130-365 days post-second vaccination. Here, we performed epitope mapping with synthetic peptides covering the whole sequence of SE36 to identify and map dominant epitopes in Ugandan adult serum presumed to have clinical immunity to P. falciparum malaria. High titer sera from the Ugandan adults predominantly reacted with peptides corresponding to two successive N-terminal regions of SERA5 containing octamer repeats and serine rich sequences, regions of SERA5 that were previously reported to have limited polymorphism. Affinity purified antibodies specifically recognizing the octamer repeats and serine rich sequences exhibited a high antibody-dependent cellular inhibition (ADCI) activity that inhibited parasite growth. Furthermore, protein structure predictions and structural analysis of SE36 using spectroscopic methods indicated that N-terminal regions possessing inhibitory epitopes are intrinsically unstructured. Collectively, these results suggest that strict tertiary structure of SE36 epitopes is not required to elicit protective antibodies in naturally immune Ugandan adults. PMID:24886718

  19. Synthesis, protein kinase inhibitory potencies, and in vitro antiproliferative activities of meridianin derivatives.

    Science.gov (United States)

    Giraud, Francis; Alves, Georges; Debiton, Eric; Nauton, Lionel; Théry, Vincent; Durieu, Emilie; Ferandin, Yoan; Lozach, Olivier; Meijer, Laurent; Anizon, Fabrice; Pereira, Elisabeth; Moreau, Pascale

    2011-07-14

    The synthesis of new meridianin derivatives is described. The indolic ring system was substituted at the C-4 to C-7 positions either by a bromine atom or by nitro or amino groups. Additionally, an iodine atom or various aryl groups were introduced at the C-5 position of the 2-aminopyrimidine ring. These compounds as well as some of their synthetic intermediates were tested for their kinase inhibitory potencies and for their in vitro antiproliferative activities. We found that this series of compounds is particularly interesting in the development of new inhibitors of DYRK1A and CLK1 kinases. The most effective compounds toward these two kinase families are the 6- and 7-bromo derivatives 30, 33, and 34 that showed more than 45-fold selectivity toward DYRK1A/CLK1 kinases over the other kinases tested. Meridianin derivatives could thus be developed toward potent and selective inhibitors of key RNA splicing regulators and potential therapeutic agents. PMID:21623630

  20. Extracellular-regulated kinase 2 is activated by the enhancement of hinge flexibility.

    OpenAIRE

    Sours, Kevin M.; Xiao,Yao; Ahn, Natalie G.

    2014-01-01

    Protein motions underlie conformational and entropic contributions to enzyme catalysis; however, relatively little is known about the ways in which this occurs. Studies of the mitogen-activated protein kinase ERK2 (extracellular-regulated protein kinase 2) by hydrogen-exchange mass spectrometry suggest that activation enhances backbone flexibility at the linker between N- and C-terminal domains while altering nucleotide binding mode. Here, we address the hypothesis that enhanced backbone flex...

  1. Structure Guided Optimization, in Vitro Activity, and in Vivo Activity of Pan-PIM Kinase Inhibitors.

    Science.gov (United States)

    Burger, Matthew T; Han, Wooseok; Lan, Jiong; Nishiguchi, Gisele; Bellamacina, Cornelia; Lindval, Mika; Atallah, Gordana; Ding, Yu; Mathur, Michelle; McBride, Chris; Beans, Elizabeth L; Muller, Kristine; Tamez, Victoriano; Zhang, Yanchen; Huh, Kay; Feucht, Paul; Zavorotinskaya, Tatiana; Dai, Yumin; Holash, Jocelyn; Castillo, Joseph; Langowski, John; Wang, Yingyun; Chen, Min Y; Garcia, Pablo D

    2013-12-12

    Proviral insertion of Moloney virus (PIM) 1, 2, and 3 kinases are serine/threonine kinases that normally function in survival and proliferation of hematopoietic cells. As high expression of PIM1, 2, and 3 is frequently observed in many human malignancies, including multiple myeloma, non-Hodgkins lymphoma, and myeloid leukemias, there is interest in determining whether selective PIM inhibition can improve outcomes of these human cancers. Herein, we describe our efforts toward this goal. The structure guided optimization of a singleton high throughput screening hit in which the potency against all three PIM isoforms was increased >10,000-fold to yield compounds with pan PIM K is < 10 pM, nanomolar cellular potency, and in vivo activity in an acute myeloid leukemia Pim-dependent tumor model is described. PMID:24900629

  2. Ack1: activation and regulation by allostery.

    Directory of Open Access Journals (Sweden)

    Ketan S Gajiwala

    Full Text Available The non-receptor tyrosine kinase Ack1 belongs to a unique multi-domain protein kinase family, Ack. Ack is the only family of SH3 domain containing kinases to have an SH3 domain following the kinase domain; others have their SH3 domains preceding the kinase domain. Previous reports have suggested that Ack1 does not require phosphorylation for activation and the enzyme activity of the isolated kinase domain is low relative to other kinases. It has been shown to dimerize in the cellular environment, which augments its enzyme activity. The molecular mechanism of activation, however, remains unknown. Here we present structural and biochemical data on Ack1 kinase domain, and kinase domain+SH3 domain that suggest that Ack1 in its monomeric state is autoinhibited, like EGFR and CDK. The activation of the kinase domain may require N-lobe mediated symmetric dimerization, which may be facilitated by the N-terminal SAM domain. Results presented here show that SH3 domain, unlike in Src family tyrosine kinases, does not directly control the activation state of the enzyme. Instead we speculate that the SH3 domain may play a regulatory role by facilitating binding of the MIG6 homologous region to the kinase domain. We postulate that features of Ack1 activation and regulation parallel those of receptor tyrosine kinase EGFR with some interesting differences.

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

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    Debora Bogani

    2009-09-01

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

  4. N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis.

    Science.gov (United States)

    Rada, Petr; Makki, Abhijith Radhakrishna; Zimorski, Verena; Garg, Sriram; Hampl, Vladimír; Hrdý, Ivan; Gould, Sven B; Tachezy, Jan

    2015-12-01

    Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PPi-dependent phosphofructokinase (TvPPi-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPPi-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to "short" bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.

  5. Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

    Energy Technology Data Exchange (ETDEWEB)

    Auperin,T.; Bolduc, G.; Baron, M.; Heroux, A.; Filman, D.; Madoff, L.; Hogle, J.

    2005-01-01

    Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis, and meningitis among neonates and an important cause of morbidity among pregnant women and immunocompromised adults. Invasive diseases due to GBS are attributed to the ability of the pathogen to translocate across human epithelial surfaces. The alpha C protein (ACP) has been identified as an invasin that plays a role in internalization and translocation of GBS across epithelial cells. The soluble N-terminal domain of ACP (NtACP) blocks the internalization of GBS. We determined the 1.86-{angstrom} resolution crystal structure of NtACP comprising residues Ser{sup 52} through Leu{sup 225} of the full-length ACP. NtACP has two domains, an N-terminal {beta}-sandwich and a C-terminal three-helix bundle. Structural and topological alignments reveal that the {beta}-sandwich shares structural elements with the type III fibronectin fold (FnIII), but includes structural elaborations that make it unique. We have identified a potential integrin-binding motif consisting of Lys-Thr-Asp{sup 146}, Arg{sup 110}, and Asp{sup 118}. A similar arrangement of charged residues has been described in other invasins. ACP shows a heparin binding activity that requires NtACP. We propose a possible heparin-binding site, including one surface of the three-helix bundle, and nearby portions of the sandwich and repeat domains. We have validated this prediction using assays of the heparin binding and cell-adhesion properties of engineered fragments of ACP. This is the first crystal structure of a member of the highly conserved Gram-positive surface alpha-like protein family, and it will enable the internalization mechanism of GBS to be dissected at the atomic level.

  6. Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator

    Directory of Open Access Journals (Sweden)

    Ayrapetov Marina K

    2005-11-01

    Full Text Available Abstract Background Protein tyrosine kinases are important enzymes for cell signalling and key targets for anticancer drug discovery. The catalytic mechanisms of protein tyrosine kinase-catalysed phosphorylation are not fully understood. Protein tyrosine kinase Csk requires two Mg2+ cations for activity: one (M1 binds to ATP, and the other (M2 acts as an essential activator. Results Experiments in this communication characterize the interaction between M2 and Csk. Csk activity is sensitive to pH in the range of 6 to 7. Kinetic characterization indicates that the sensitivity is not due to altered substrate binding, but caused by the sensitivity of M2 binding to pH. Several residues in the active site with potential of binding M2 are mutated and the effect on metal activation studied. An active mutant of Asn319 is generated, and this mutation does not alter the metal binding characteristics. Mutations of Glu236 or Asp332 abolish the kinase activity, precluding a positive or negative conclusion on their role in M2 coordination. Finally, the ability of divalent metal cations to activate Csk correlates to a combination of ionic radius and the coordination number. Conclusion These studies demonstrate that M2 binding to Csk is sensitive to pH, which is mainly responsible for Csk activity change in the acidic arm of the pH response curve. They also demonstrate critical differences in the metal activator coordination sphere in protein tyrosine kinase Csk and a protein Ser/Thr kinase, the cAMP-dependent protein kinase. They shed light on the physical interactions between a protein tyrosine kinase and a divalent metal activator.

  7. PERK Utilizes Intrinsic Lipid Kinase Activity To Generate Phosphatidic Acid, Mediate Akt Activation, and Promote Adipocyte Differentiation

    Science.gov (United States)

    Bobrovnikova-Marjon, Ekaterina; Pytel, Dariusz; Riese, Matthew J.; Vaites, Laura Pontano; Singh, Nickpreet; Koretzky, Gary A.; Witze, Eric S.

    2012-01-01

    The endoplasmic reticulum (ER) resident PKR-like kinase (PERK) is necessary for Akt activation in response to ER stress. We demonstrate that PERK harbors intrinsic lipid kinase, favoring diacylglycerol (DAG) as a substrate and generating phosphatidic acid (PA). This activity of PERK correlates with activation of mTOR and phosphorylation of Akt on Ser473. PERK lipid kinase activity is regulated in a phosphatidylinositol 3-kinase (PI3K) p85α-dependent manner. Moreover, PERK activity is essential during adipocyte differentiation. Because PA and Akt regulate many cellular functions, including cellular survival, proliferation, migratory responses, and metabolic adaptation, our findings suggest that PERK has a more extensive role in insulin signaling, insulin resistance, obesity, and tumorigenesis than previously thought. PMID:22493067

  8. PERK utilizes intrinsic lipid kinase activity to generate phosphatidic acid, mediate Akt activation, and promote adipocyte differentiation.

    Science.gov (United States)

    Bobrovnikova-Marjon, Ekaterina; Pytel, Dariusz; Riese, Matthew J; Vaites, Laura Pontano; Singh, Nickpreet; Koretzky, Gary A; Witze, Eric S; Diehl, J Alan

    2012-06-01

    The endoplasmic reticulum (ER) resident PKR-like kinase (PERK) is necessary for Akt activation in response to ER stress. We demonstrate that PERK harbors intrinsic lipid kinase, favoring diacylglycerol (DAG) as a substrate and generating phosphatidic acid (PA). This activity of PERK correlates with activation of mTOR and phosphorylation of Akt on Ser473. PERK lipid kinase activity is regulated in a phosphatidylinositol 3-kinase (PI3K) p85α-dependent manner. Moreover, PERK activity is essential during adipocyte differentiation. Because PA and Akt regulate many cellular functions, including cellular survival, proliferation, migratory responses, and metabolic adaptation, our findings suggest that PERK has a more extensive role in insulin signaling, insulin resistance, obesity, and tumorigenesis than previously thought.

  9. Mitogen-activated protein kinase-dependent apoptosis in norcan-tharidin-treated A375-S2 cells is proceeded by the activation of protein kinase C

    Institute of Scientific and Technical Information of China (English)

    AN Wei-wei; WANG Min-wei; Tashiro Shin-ichi; Onodera Satoshi; Ikejima Takashi

    2005-01-01

    Background We have reported that norcantharidin (NCTD) induces human melanoma A375-S2 cell apoptosis and that the activation of caspase and the mitochondrial pathway are involved in the apoptotic process. This study aimed at investigating the roles of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) in A375-S2 cell apoptosis induced by NCTD. Methods We assessed the effects of NCTD on cell growth inhibition using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay, DNA fragmentation (DNA agarose gel electrophoresis), and MAPK protein levels (Western blot analysis) in A375-S2 cells. Photomicroscopic data were also collected.Results The NCTD inhibitory effect on A375-S2 cells was partially reversed by MAPK and PKC inhibitors. The expression of phosphorylated JNK and p38 also increased after the treatment with NCTD, and inhibitors of c-Jun NH2-terminal kinase (JNK) and p38 (SP600125 and SB203580, respectively) had significant inhibitory effects on the upregulation of phosphorylated JNK and p38 expression. Simultaneously, the PKC inhibitor staurosporine blocked the upregulation of phosphorylated JNK and phosphorylated p38, but had little effect on extracellular signal-regulated kinase (ERK) expression. Conclusion These results suggest that the activation of JNK and p38 MAPK promotes the process of NCTD-induced A375-S2 cell apoptosis and that PKC plays an important regulation role in the activation of MAPKs.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-08-01

    Activation of calcium-activated, phospholipid-dependent protein kinase C by diacylglycerol and phorbol esters has been shown to mediate release of hormones in many systems. To determine whether protein kinase C activation is also involved in the regulation of prolactin release from human decidual, the authors have examined the effects of various acylglycerols and phorbol esters on the synthesis and release of prolactin from cultured human decidual cells. sn-1,2-Dioctanolyglycerol (diC8), which is known to stimulate protein kinase C in other systems, inhibited prolactin release in a dose-dependent manner with maximal inhibition of 53.1% at 100 M. Diolein (100 M), which also stimulates protein kinase C activity in some systems, inhibited prolactin release by 21.3%. Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 4US -phorbol 12,13-dibutyrate, which activate protein kinase C in other systems, also inhibited the release of prolactin, which the protein kinase C inactivate 4 -phorbol-12,13-didecanoate was without effect. The inhibition of prolactin release was secondary to a decrease in prolactin synthesis. Although diC8 and PMA inhibited the synthesis and release of prolactin, these agents had no effect on the synthesis or release of trichloroacetic acid-precipitable (TVS)methionine-labeled decidual proteins and did not cause the release of the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. DiC8 and PMA stimulates the specific activity of protein kinase C in decidual tissue by 14.6 and 14.0-fold, respectively. The inhibition of the synthesis and release of prolactin by diC8 and phorbol esters strongly implicates protein kinase C in the regulation of the production and release of prolactin from the decidua.

  11. Predicting kinase activity in angiotensin receptor phosphoproteomes based on sequence-motifs and interactions.

    Directory of Open Access Journals (Sweden)

    Rikke Bøgebo

    Full Text Available Recent progress in the understanding of seven-transmembrane receptor (7TMR signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT1aR is one of the most studied 7TMRs with respect to selective activation of the β-arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data. The method builds upon NetworKIN, which applies sophisticated linear motif analysis in combination with contextual network modelling to predict kinase-substrate associations with high accuracy and sensitivity. These predictions form the basis for subsequently nonparametric statistical analysis to identify likely activated kinases. This suggested that AT1aR-dependent signalling activates 48 of the 285 kinases detected in HEK293 cells. Of these, Aurora B, CLK3 and PKG1 have not previously been described in the pathway whereas others, such as PKA, PKB and PKC, are well known. In summary, we have developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets.

  12. Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

    Directory of Open Access Journals (Sweden)

    Hou Ssu-Yu

    2010-06-01

    Full Text Available Abstract Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors (statins have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin. Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2 formation, and phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP phosphorylation, and endothelial nitric oxide synthase (eNOS expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP

  13. Distinct Mechanisms of Receptor and Nonreceptor Tyrosine Kinase Activation by Reactive Oxygen Species in Vascular Smooth Muscle Cells: Role of Metalloprotease and Protein Kinase C-δ

    OpenAIRE

    Frank, Gerald D.; Mifune, Mizuo; Inagami, Tadashi; Ohba, Motoi; Sasaki, Terukatsu; Higashiyama, Shigeki; Dempsey, Peter J; Eguchi, Satoru

    2003-01-01

    Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor...

  14. Structural Insights into the HWE Histidine Kinase Family: The Brucella Blue Light-Activated Histidine Kinase Domain.

    Science.gov (United States)

    Rinaldi, Jimena; Arrar, Mehrnoosh; Sycz, Gabriela; Cerutti, María Laura; Berguer, Paula M; Paris, Gastón; Estrín, Darío Ariel; Martí, Marcelo Adrián; Klinke, Sebastián; Goldbaum, Fernando Alberto

    2016-03-27

    In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK.

  15. AMP-activated protein kinase phosphorylation in brain is dependent on method of sacrifice and tissue preparation

    OpenAIRE

    Scharf, Matthew T.; Mackiewicz, Miroslaw; Naidoo, Nirinjini; O'Callaghan, James P.; Pack, Allan I.

    2007-01-01

    AMP-activated protein kinase is activated when the catalytic α subunit is phosphorylated on Thr172 and therefore, phosphorylation of the α subunit is used as a measure of activation. However, measurement of α-AMP-activated protein kinase phosphorylation in vivo can be technically challenging. To determine the most accurate method for measuring α-AMP-activated protein kinase phosphorylation in the mouse brain, we compared different methods of sacrifice and tissue preparation. We found that fre...

  16. Protective effects of inhibition of adenosine monophosphate activated protein kinase activity against cerebral ischemia-reperfusion injury in mice

    Institute of Scientific and Technical Information of China (English)

    补娟

    2013-01-01

    Objective To observe the effect of inhibition of adenosine monophosphate activated protein kinase (AMPK) on shape,function and inflammatory factor of microglia for mice after cerebral ischemia-reperfusion

  17. FAK kinase activity is required for the progression of c-Met/β-catenin-driven HCC

    Science.gov (United States)

    Shang, Na; Arteaga, Maribel; Zaidi, Ali; Cotler, Scott J.; Breslin, Peter; Ding, Xianzhong; Kuo, Paul; Nishimura, Michael; Zhang, Jiwang; Qiu, Wei

    2016-01-01

    Background & Aims There is an urgent need to develop new and more effective therapeutic strategies and agents to treat hepatocellular carcinoma (HCC). We have recently found that deletion of Fak in hepatocytes before tumors form inhibits tumor development and prolongs survival of animals in a c-Met (MET)/β-catenin (CAT)-driven HCC mouse model. However, it has yet to be determined whether FAK expression in hepatocytes promotes MET/CAT-induced HCC progression after tumor initiation. In addition, it remains unclear whether FAK promotes HCC development through its kinase activity. Methods We generated hepatocyte-specific inducible Fak-deficient mice (Alb-creERT2; Fakflox/flox) to examine the role of FAK in HCC progression. We re-expressed wild-type and mutant FAK in Fak-deficient mice to determine FAK’s kinase activity in HCC development. We also examined the efficacy of a FAK kinase inhibitor PF-562271 on HCC inhibition. Results We found that deletion of Fak after tumors form significantly repressed MET/CAT-induced tumor progression. Ectopic FAK expression restored HCC formation in hepatocyte-specific Fak-deficient mice. However, overexpression of a FAK kinase-dead mutant led to reduced tumor load compared to mice which express wild-type FAK. Furthermore, PF-562271 significantly suppressed progression of MET/CAT-induced HCC. Conclusion Fak kinase activity is important for MET/CAT-induced HCC progression. Inhibiting FAK kinase activity provides a potential therapeutic strategy to treat HCC. PMID:27142958

  18. RNA-dependent protein kinase (PKR) depletes nutrients, inducing phosphorylation of AMP-activated kinase in lung cancer.

    Science.gov (United States)

    Guo, Chengcheng; Hao, Chuncheng; Shao, RuPing; Fang, Bingliang; Correa, Arlene M; Hofstetter, Wayne L; Roth, Jack A; Behrens, Carmen; Kalhor, Neda; Wistuba, Ignacio I; Swisher, Stephen G; Pataer, Apar

    2015-05-10

    We have demonstrated that RNA-dependent protein kinase (PKR) and its downstream protein p-eIF2α are independent prognostic markers for overall survival in lung cancer. In the current study, we further investigate the interaction between PKR and AMPK in lung tumor tissue and cancer cell lines. We examined PKR protein expression in 55 frozen primary lung tumor tissues by Western blotting and analyzed the association between PKR expression and expression of 139 proteins on tissue samples examined previously by Reverse Phase Protein Array (RPPA) from the same 55 patients. We observed that biomarkers were either positively (phosphorylated AMP-activated kinase(T172) [p-AMPK]) or negatively (insulin receptor substrate 1, meiotic recombination 11, ATR interacting protein, telomerase, checkpoint kinase 1, and cyclin E1) correlated with PKR. We further confirmed that induction of PKR with expression vectors in lung cancer cells causes activation of the AMPK protein independent of the LKB1, TAK1, and CaMKKβ pathway. We found that PKR causes nutrient depletion, which increases AMP levels and decreases ATP levels, causing AMPK phosphorylation. We further demonstrated that inhibiting AMPK expression with compound C or siRNA enhanced PKR-mediated cell death. We next explored the combination of PKR and p-AMPK expression in NSCLC patients and observed that expression of p-AMPK predicted a poor outcome for adenocarcinoma patients with high PKR expression and a better prognosis for those with low PKR expression. These findings were consistent with our in vitro results. AMPK might rescue cells facing metabolic stresses, such as ATP depletion caused by PKR. Our data indicate that PKR causes nutrient depletion, which induces the phosphorylation of AMPK. AMPK might act as a protective response to metabolic stresses, such as nutrient deprivation. PMID:25798539

  19. Sphingosine induces phospholipase D and mitogen activated protein kinase in vascular smooth muscle cells.

    Science.gov (United States)

    Taher, M M; Abd-Elfattah, A S; Sholley, M M

    1998-12-01

    The enzymes phospholipase D and diacylglycerol kinase generate phosphatidic acid which is considered to be a mitogen. Here we report that sphingosine produced a significant amount of phosphatidic acid in vascular smooth muscle cells from the rat aorta. The diacylglycerol kinase inhibitor R59 949 partially depressed sphingosine induced phosphatidic acid formation, suggesting that activation of phospholipase C and diacylglycerol kinase can not account for the bulk of phosphatidic acid produced and that additional pathways such as phospholipase D may contribute to this. Further, we have shown that phosphatidylethanol was produced by sphingosine when vascular smooth muscle cells were stimulated in the presence of ethanol. Finally, as previously shown for other cell types, sphingosine stimulated mitogen-activated protein kinase in vascular smooth muscle cells.

  20. Calcium-Dependent Protein Kinase CPK21 Functions in Abiotic Stress Response in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Sandra Franz; Britta Ehlert; Anja Liese; Joachim Kurth; Anne-Claire Cazalé; Tina Romeis

    2011-01-01

    Calcium-dependent protein kinases(CDPKs)comprise a family of plant serine/threonine protein kinases in which the calcium sensing domain and the kinase effector domain are combined within one molecule.So far,a biological function in abiotic stress signaling has only been reported for few CDPK isoforms,whereas the underlying biochemical mechanism for these CDPKs is still mainly unknown.Here,we show that CPK21 from Arabidopsis thaliana is biochemically activated in vivo in response to hyperosmotic stress.Loss-of-function seedlings of cpk21 are more tolerant to hyperosmotic stress and mutant plants show increased stress responses with respect to marker gene expression and metabolite accumulation.In transgenic Arabidopsis complementation lines in the cpk21 mutant background,in which either CPK21 wildtype,or a full-length enzyme variant carrying an amino-acid substitution were stably expressed,stress responsitivity was restored by CPK21 but not with the kinase inactive variant.The biochemical characterization of in planta synthesized and purified CPK21 protein revealed that within the calcium-binding domain,N-terminal EF1- and EF2-motifs compared to C-terminal EF3- and EF4-motifs differ in their contribution to calcium-regulated kinase activity,suggesting a crucial role for the N-terminal EF-hand pair.Our data provide evidence for CPK21 contributing in abiotic stress signaling and suggest that the N-terminal EF-hand pair is a calcium-sensing determinant controlling specificity of CPK21 function.

  1. Association between mitogen-activated protein kinase kinase kinase 1 polymorphisms and breast cancer susceptibility: a meta-analysis of 20 case-control studies.

    Directory of Open Access Journals (Sweden)

    Qiaoli Zheng

    Full Text Available BACKGROUND: The genome-wide single-nucleotide polymorphisms (SNPs profiles can be used as diagnostic markers for human cancers. The associations between mitogen-activated protein kinase kinase kinase 1 (MAP3K1 SNPs rs889312 A>C, rs16886165 T>G and breast cancer risk have been widely evaluated, but the results were inconsistent. To derive a conclusive assessment of the associations, we performed a meta-analysis by combining data from all eligible case-control studies up to date. METHODS: By searching PubMed, ISI web of knowledge, Embase and Cochrane databases, we identified all eligible studies published before September 2013. Odds ratios (ORs with 95% confidence intervals (CIs were used to assess the strength of associations in fixed-effect or random-effect model. False-positive report probability (FPRP was calculated to confirm the significance of the results. RESULTS: A total of 59670 cases in 20 case-control studies were included in this meta-analysis. Significant associations with breast cancer risk were observed for SNPs rs889312 and rs16886165 polymorphisms with a per-allele OR of 1.11 (95% CI: 1.09-1.13 and 1.14 (95% CI: 1.09-1.20 respectively. For rs889312, in subgroup analysis by ethnicity, significant associations were identified in Europeans and Asians, but not in Africans. When stratified by estrogen receptor (ER expression status, rs889312 was associated with both ER-positive and ER-negative breast cancers. Results from the FPRP analyses were consistent with and supportive to the above results. CONCLUSIONS: The present meta-analysis suggests that rs889312-C allele and rs16886165-G allele might be risk factors for breast cancer, especially in Europeans and Asians.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-11-15

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

  3. AMP-activated protein kinase downregulates Kv7.1 cell surface expression

    DEFF Research Database (Denmark)

    Andersen, Martin N; Krzystanek, Katarzyna; Jespersen, Thomas;

    2012-01-01

    in response to polarization of the epithelial Madin-Darby canine kidney (MDCK) cell line and that this was mediated by activation of protein kinase C (PKC). In this study, the pathway downstream of PKC, which leads to internalization of Kv7.1 upon cell polarization, is elucidated. We show by confocal...... microscopy that Kv7.1 is endocytosed upon initiation of the polarization process and sent for degradation by the lysosomal pathway. The internalization could be mimicked by pharmacological activation of the AMP-activated protein kinase (AMPK) using three different AMPK activators. We demonstrate...

  4. Method of empirical dependences in estimation and prediction of activity of creatine kinase isoenzymes in cerebral ischemia

    Science.gov (United States)

    Sergeeva, Tatiana F.; Moshkova, Albina N.; Erlykina, Elena I.; Khvatova, Elena M.

    2016-04-01

    Creatine kinase is a key enzyme of energy metabolism in the brain. There are known cytoplasmic and mitochondrial creatine kinase isoenzymes. Mitochondrial creatine kinase exists as a mixture of two oligomeric forms - dimer and octamer. The aim of investigation was to study catalytic properties of cytoplasmic and mitochondrial creatine kinase and using of the method of empirical dependences for the possible prediction of the activity of these enzymes in cerebral ischemia. Ischemia was revealed to be accompanied with the changes of the activity of creatine kinase isoenzymes and oligomeric state of mitochondrial isoform. There were made the models of multiple regression that permit to study the activity of creatine kinase system in cerebral ischemia using a calculating method. Therefore, the mathematical method of empirical dependences can be applied for estimation and prediction of the functional state of the brain by the activity of creatine kinase isoenzymes in cerebral ischemia.

  5. RKIP regulates MAP kinase signaling in cells with defective B-Raf activity.

    Science.gov (United States)

    Zeng, Lingchun; Ehrenreiter, Karin; Menon, Jyotsana; Menard, Ray; Kern, Florian; Nakazawa, Yoko; Bevilacqua, Elena; Imamoto, Akira; Baccarini, Manuela; Rosner, Marsha Rich

    2013-05-01

    MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf kinase inhibitory protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism. Loss of B-Raf protein or activity significantly reduces MAPK activation in these cells. We show that RKIP depletion can rescue the compromised ERK activation and promote proliferation, and this rescue occurs through a Raf-1 dependent mechanism. These results provide formal evidence that RKIP is a bona fide regulator of Raf-1. We propose a new model in which RKIP plays a key role in regulating the ability of cells to signal through Raf-1 to ERK in B-Raf compromised cells.

  6. Crystal structure of the kinase domain of serum and glucocorticoid-regulated kinase 1 in complex with AMP–PNP

    Science.gov (United States)

    Zhao, Baoguang; Lehr, Ruth; Smallwood, Angela M.; Ho, Thau F.; Maley, Kathleen; Randall, Tanya; Head, Martha S.; Koretke, Kristin K.; Schnackenberg, Christine G.

    2007-01-01

    Serum and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine protein kinase of the AGC family which participates in the control of epithelial ion transport and is implicated in proliferation and apoptosis. We report here the 1.9 Å crystal structure of the catalytic domain of inactive human SGK1 in complex with AMP–PNP. SGK1 exists as a dimer formed by two intermolecular disulfide bonds between Cys258 in the activation loop and Cys193. Although most of the SGK1 structure closely resembles the common protein kinase fold, the structure around the active site is unique when compared to most protein kinases. The αC helix is not present in this inactive form of SGK1 crystal structure; instead, the segment corresponding to the C helix forms a β-strand that is stabilized by the N-terminal segment of the activation loop through a short antiparallel β-sheet. Since the differences from other kinases occur around the ATP binding site, this structure can provide valuable insight into the design of selective and highly potent ATP-competitive inhibitors of SGK1 kinase. PMID:17965184

  7. Crystal structure of the kinase domain of serum and glucocorticoid-regulated kinase 1 in complex with AMP-PNP

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Baoguang; Lehr, Ruth; Smallwood, Angela M; Ho, Thau F; Maley, Kathleen; Randall, Tanya; Head, Martha S; Koretke, Kristin K; Schnackenberg, Christine G [GSKPA

    2008-06-30

    Serum and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine protein kinase of the AGC family which participates in the control of epithelial ion transport and is implicated in proliferation and apoptosis. We report here the 1.9 {angstrom} crystal structure of the catalytic domain of inactive human SGK1 in complex with AMP-PNP. SGK1 exists as a dimer formed by two intermolecular disulfide bonds between Cys258 in the activation loop and Cys193. Although most of the SGK1 structure closely resembles the common protein kinase fold, the structure around the active site is unique when compared to most protein kinases. The {alpha}C helix is not present in this inactive form of SGK1 crystal structure; instead, the segment corresponding to the C helix forms a {beta}-strand that is stabilized by the N-terminal segment of the activation loop through a short antiparallel {beta}-sheet. Since the differences from other kinases occur around the ATP binding site, this structure can provide valuable insight into the design of selective and highly potent ATP-competitive inhibitors of SGK1 kinase.

  8. Function of the N-terminal segment of the RecA-dependent nuclease Ref.

    Science.gov (United States)

    Gruber, Angela J; Olsen, Tayla M; Dvorak, Rachel H; Cox, Michael M

    2015-02-18

    The bacteriophage P1 Ref (recombination enhancement function) protein is a RecA-dependent, HNH endonuclease. It can be directed to create targeted double-strand breaks within a displacement loop formed by RecA. The 76 amino acid N-terminal region of Ref is positively charged (25/76 amino acid residues) and inherently unstructured in solution. Our investigation of N-terminal truncation variants shows this region is required for DNA binding, contains a Cys involved in incidental dimerization and is necessary for efficient Ref-mediated DNA cleavage. Specifically, Ref N-terminal truncation variants lacking between 21 and 47 amino acids are more effective RecA-mediated targeting nucleases. We propose a more refined set of options for the Ref-mediated cleavage mechanism, featuring the N-terminal region as an anchor for at least one of the DNA strand cleavage events.

  9. Phosphorylation by PKA potentiates retinoic acid receptor α activity by means of increasing interaction with and phosphorylation by cyclin H/cdk7

    OpenAIRE

    Gaillard, Emilie; Bruck, Nathalie; Brelivet, Yann; Bour, Gaétan; Lalevée, Sébastien; Bauer, Annie; Poch, Olivier; Moras, Dino; Rochette-Egly, Cécile

    2006-01-01

    Nuclear retinoic acid receptors (RARs) work as ligand-dependent heterodimeric RAR/retinoid X receptor transcription activators, which are targets for phosphorylations. The N-terminal activation function (AF)-1 domain of RARα is phosphorylated by the cyclin-dependent kinase (cdk) 7/cyclin H complex of the general transcription factor TFIIH and the C-terminal AF-2 domain by the cAMP-dependent protein kinase A (PKA). Here, we report the identification of a molecular pathway by which phosphorylat...

  10. Comprehensive Characterization of AMP-Activated Protein Kinase Catalytic Domain by Top-Down Mass Spectrometry

    Science.gov (United States)

    Yu, Deyang; Peng, Ying; Ayaz-Guner, Serife; Gregorich, Zachery R.; Ge, Ying

    2016-02-01

    AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ). C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ had noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems.

  11. Mutations that cause threonine sensitivity identify catalytic and regulatory regions of the aspartate kinase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Arévalo-Rodríguez, M; Calderón, I L; Holmberg, S

    1999-01-01

    . The corresponding amino acid substitutions, K26I and G25D, affect residues located in the vicinity of a highly conserved lysine-phenylalanine-glycine-glycine (KFGG) stretch present in the N-terminal part of the aspartate kinase, to which no function has so far been assigned. We suggest that this region is involved...... inhibited by threonine than the wild-type enzyme. The predicted amino acid substitution in this mutant, A406T, is located in a region associated with the modulation of the enzymatic activity. The other two mutants carry an aspartate kinase with reduced affinity for its substrates, aspartate and ATP...

  12. The methylated N-terminal tail of RCC1 is required for stabilisation of its interaction with chromatin by Ran in live cells

    Directory of Open Access Journals (Sweden)

    Sanderson Helen S

    2010-06-01

    Full Text Available Abstract Background Regulator of chromosome condensation 1 (RCC1 is the guanine nucleotide exchange factor for Ran GTPase. Localised generation of Ran-GTP by RCC1 on chromatin is critical for nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Both the N-terminal tail of RCC1 and its association with Ran are important for its interaction with chromatin in cells. In vitro, the association of Ran with RCC1 induces a conformational change in the N-terminal tail that promotes its interaction with DNA. Results We have investigated the mechanism of the dynamic interaction of the α isoform of human RCC1 (RCC1α with chromatin in live cells using fluorescence recovery after photobleaching (FRAP of green fluorescent protein (GFP fusions. We show that the N-terminal tail stabilises the interaction of RCC1α with chromatin and this function can be partially replaced by another lysine-rich nuclear localisation signal. Removal of the tail prevents the interaction of RCC1α with chromatin from being stabilised by RanT24N, a mutant that binds stably to RCC1α. The interaction of RCC1α with chromatin is destabilised by mutation of lysine 4 (K4Q, which abolishes α-N-terminal methylation, and this interaction is no longer stabilised by RanT24N. However, α-N-terminal methylation of RCC1α is not regulated by the binding of RanT24N. Conversely, the association of Ran with precipitated RCC1α does not require the N-terminal tail of RCC1α or its methylation. The mobility of RCC1α on chromatin is increased by mutation of aspartate 182 (D182A, which inhibits guanine-nucleotide exchange activity, but RCC1αD182A can still bind nucleotide-free Ran and its interaction with chromatin is stabilised by RanT24N. Conclusions These results show that the stabilisation of the dynamic interaction of RCC1α with chromatin by Ran in live cells requires the N-terminal tail of RCC1α. α-N-methylation is not regulated by formation of the binary

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

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Kap; Pullalarevu, Sadhana; Surabian, Karen Talin; Howard, Andrew; Suzuki, Tomohiko; Moult, John; Herzberg, Osnat (Kochi); (IIT); (UMBI)

    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 longer 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 optimized once

  14. Resveratrol inhibits Trypanosoma cruzi arginine kinase and exerts a trypanocidal activity.

    Science.gov (United States)

    Valera Vera, Edward A; Sayé, Melisa; Reigada, Chantal; Damasceno, Flávia S; Silber, Ariel M; Miranda, Mariana R; Pereira, Claudio A

    2016-06-01

    Arginine kinase catalyzes the reversible transphosphorylation between ADP and phosphoarginine which plays a critical role in the maintenance of cellular energy homeostasis. Arginine kinase from the protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, meets the requirements to be considered as a potential therapeutic target for rational drug design including being absent in its mammalian hosts. In this study a group of polyphenolic compounds was evaluated as potential inhibitors of arginine kinase using molecular docking techniques. Among the analyzed compounds with the lowest free binding energy to the arginine kinase active site (market price; and (3) has as a well-defined target enzyme which is absent in the mammalian host, it is a promising compound as a trypanocidal drug for Chagas disease. PMID:26976067

  15. Autophosphorylation of serine 608 in the p85 regulatory subunit of wild type or cancer-associated mutants of phosphoinositide 3-kinase does not affect its lipid kinase activity

    OpenAIRE

    Layton Meredith J; Saad Mirette; Church Nicole L; Pearson Richard B; Mitchell Christina A; Phillips Wayne A

    2012-01-01

    Abstract Background The α-isoform of the Type 1A Phosphoinositide 3-kinases (PI3Kα) has protein kinase activity as well as phosphoinositide lipid kinase activity. The best described substrate for its protein kinase activity is its regulatory subunit, p85α, which becomes phosphorylated on Serine 608. Phosphorylation of Serine 608 has been reported to down-regulate its lipid kinase activity. Results We have assessed whether oncogenic mutants of PI3Kα, which have up-regulated lipid kinase activi...

  16. Extracellular signal-regulated kinases 1 and 2 activation in endothelial cells exposed to cyclic strain

    Science.gov (United States)

    Ikeda, M.; Takei, T.; Mills, I.; Kito, H.; Sumpio, B. E.

    1999-01-01

    The aim of this study was to determine whether extracellular signal-regulated kinases 1/2 (ERK1/ERK2) are activated and might play a role in enhanced proliferation and morphological change induced by strain. Bovine aortic endothelial cells (BAEC) were subjected to an average of 6 or 10% strain at a rate of 60 cycles/min for up to 4 h. Cyclic strain caused strain- and time-dependent phosphorylation and activation of ERK1/ERK2. Peak phosphorylation and activation of ERK1/ERK2 induced by 10% strain were at 10 min. A specific ERK1/ERK2 kinase inhibitor, PD-98059, inhibited phosphorylation and activation of ERK1/ERK2 but did not inhibit the increased cell proliferation and cell alignment induced by strain. Treatment of BAEC with 2,5-di-tert-butyl-1, 4-benzohydroquinone, to deplete inositol trisphosphate-sensitive calcium storage, and gadolinium chloride, a Ca2+ channel blocker, did not inhibit the activation of ERK1/ERK2. Strain-induced ERK1/ERK2 activation was partly inhibited by the protein kinase C inhibitor calphostin C and completely inhibited by the tyrosine kinase inhibitor genistein. These data suggest that 1) ERK1/ERK2 are not critically involved in the strain-induced cell proliferation and orientation, 2) strain-dependent activation of ERK1/ERK2 is independent of intracellular and extracellular calcium mobilization, and 3) protein kinase C activation and tyrosine kinase regulate strain-induced activation of ERK1/ERK2.

  17. Diglyceride kinase activity of microtubules. Characterization and comparison with the protein kinase and ATPase activities associated with vinblastine-isolated tubulin of chick embryonic muscles.

    Science.gov (United States)

    Daleo, G R; Piras, M M; Piras, R

    1976-09-15

    Vinblastine-isolated microtubule protein from chick embryonic muscles has an enzymatic activity which catalyzes the formation of phosphatidic acid from diglycerides and ATP. The pH optimum (6.4), sedimentation on sucrose gradients (Mr = 85 000), and sensitivity to ions of this diglyceride kinase activity are different to those of a similar enzymatic activity present in 150 000 X g supernatants of chick embryonic muscle homogenates, suggesting that it is a different species which is associated specifically with the microtubules. The reaction requires a divalent ion (e.g. 0.4 mM Mg2+ gives half-maximal stimulation), and GTP can replace ATP rather effectively, especially at nucleotide concentrations lower than 50 muM. The sedimentation of the diglyceride kinase on sucrose gradients coincides with that of the microtubules-associated protein kinase (Mr = 75 000); the heat-stability and sensivitity to proteolysis of both activities are also very similar. Stimulation of one reaction by the addition of the corresponding exogenous substrate does not impair the phosphorylation of the other, and no radioactivity is lost from phosphatidic acid or the protein moiety upon incubation of pre-labelled microtubules with a large excess of unlabelled ATP or GTP. In addition to diglyceride and protein kinase activities (0.2 and 0.3 nmol 32P-transferred X min-1 X mg-1 microtubular protein, respectively), microtubules also contain an associated ATPase (2.8 nmol X min-1 X mg-1), which requires either Mg2+ or Ca2+, can hydrolyze GTP quite effectively, and sediments with a molecular weight of 95000. The results obtained are discussed in connection with the possible relationships existing among these enzymatic activities, as well as their probable role in microtubular functions.

  18. Effect of cigarette smoke extract on lipopolysaccha-ride-activated mitogen-activated protein kinase signal transduction pathway in cultured cells

    Institute of Scientific and Technical Information of China (English)

    LI Wen; XU Yong-jian; SHEN Hua-hao

    2007-01-01

    Background Lipopolysaccharide (LPS) forms outer membrane of the wall of Gram-negative cells. LPS can directly cause damage to epithelia of respiratory tract and is the major factor responsible for the chronic inflammation of respiratory passage. The mitogen-activated protein kinase (MAPK) signal transduction pathway of the airway epithelia is intimately associated with the action of LPS. The chronic inflammation of respiratory tract and smoking are interrelated and entwined in the development and progression of chronic lung diseases. This study was designed to examine the effects of cigarette smoke extract (CSE) and LPS on MAPK signal transduction pathway in order to further understand the roles CSE and LPS play in chronic lung inflammation.Methods Cultured primary human epithelial cells of airway were divided into four groups according to the stimulants used: blank control group, LPS-stimulation group, CSE-stimulation group and CSE plus LPS group. Western blotting was employed for the detection of phosphorylation level of extracellular-signal-regulated-kinase (ERK1/2), p38 MAPK and c-Jun N-terminal kinase (JNK). The expression of cytokines of MAPK transduction pathway (granulocyte-macrophage colony stimulating factor (GM-CSF) and mRNA of IL-8) in the primary epithelial cells of respiratory tract was also determined.Results Western blotting revealed that the phosphorylation levels of ERK1/2, p38 MAPK and JNK were low and 2 hours after the LPS stimulation, the phosphorylation of ERK1/2, p38 MAPK and JNK were all increased. There was a significant difference in the phosphorylation between the LPS-stimulation group and blank control group (P<0.05); no significant difference was found between CSE-stimulation group and blank control group (P>0.05); there was a significant difference between CSE + LPS group and blank control group and between CSE + LPS group and LPS group (P<0.05). The phosphorylation of CSE-LPS group was higher than that of blank control group but

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    Activation of the ATR kinase following perturbations to DNA replication relies on a complex mechanism involving ATR recruitment to RPA-coated single-stranded DNA via its binding partner ATRIP and stimulation of ATR kinase activity by TopBP1. Here, we discovered an independent ATR activation pathway...... in vertebrates, mediated by the uncharacterized protein ETAA1 (Ewing's tumour-associated antigen 1). Human ETAA1 accumulates at DNA damage sites via dual RPA-binding motifs and promotes replication fork progression and integrity, ATR signalling and cell survival after genotoxic insults. Mechanistically......, this requires a conserved domain in ETAA1 that potently and directly stimulates ATR kinase activity independently of TopBP1. Simultaneous loss of ETAA1 and TopBP1 gives rise to synthetic lethality characterized by massive genome instability and abrogation of ATR-dependent signalling. Our findings demonstrate...

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

    Energy Technology Data Exchange (ETDEWEB)

    Nagamine, Tadashi; Nomada, Shohgo; Onouchi, Takashi; Kameshita, Isamu; Sueyoshi, Noriyuki, E-mail: sueyoshi@ag.kagawa-u.ac.jp

    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 a 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.

  1. Corticosterone activates Erk1/2 mitogen-activated protein kinase in primary hippocampal cells through rapid nongenomic mechanism

    Institute of Scientific and Technical Information of China (English)

    QI Aiqun; QIU Jian; XIAO Lin; CHEN Yizhang

    2005-01-01

    Nongenomic effects of glucocorticoids (GC) in various cell types have been well documented, but it still remains unknown whether the mechanism also works in hippocampus which is a crucial target of glucocorticoids in neural system during physiological and/or pathophysiological processes. We present here that corticosterone (B) could rapidly activate Erk1/2 mitogen-activated protein kinase (MAPK) in primarily cultured hippocampal cells within minutes, with a bell-shaped time dependent curve which peaked at 15min and then went down to normal level in 30 min. This activation was blocked by protein kinase C (PKC) inhibitor (Go6976), G protein inhibitor (GDPβs), and MEK(MAPK/extracellular signal-regulated kinase kinase) inhibitor(PD98059), but not by protein kinase A (PKA) inbibitor (H89), tyrosine kinase inhibitor (genistein), and glucocorticoid receptor ( GR ) antagonist (RU38486). Thus, the rapid activation of Erk1/2 MAPK in primary hippocampal cells induced by B was likely mediated by a G protein coupled receptor (GPCR) pathway with involvement of PKC, which belonged to the nongenomic rather than genomic mechanism of GC' s effects.

  2. AMP-activated protein kinase is activated by non-steroidal anti-inflammatory drugs.

    Science.gov (United States)

    King, Tanya S; Russe, Otto Quintus; Möser, Christine V; Ferreirós, Nerea; Kynast, Katharina L; Knothe, Claudia; Olbrich, Katrin; Geisslinger, Gerd; Niederberger, Ellen

    2015-09-01

    AMP-activated kinase (AMPK) is a cellular energy sensor, which is activated in stages of increased adenosine triphosphate (ATP) consumption. Its activation has been associated with a number of beneficial effects such as decrease of inflammatory processes and inhibition of disease progression of diabetes and obesity. A recent study suggested that salicylate, the active metabolite of the non-steroidal anti-inflammatory drug (NSAID) acetyl-salicylic acid (aspirin), is able to activate AMPK pharmacologically. This observation raised the question whether or not other NSAIDs might also act as AMPK activators and whether this action might contribute to their cyclooxygenase (COX)-independent anti-inflammatory properties. In this study, we investigated mouse and human neuronal cells and liver tissue of mice after treatment with various NSAIDs. Our results showed that the non-selective acidic NSAIDs ibuprofen and diclofenac induced AMPK activation similar to aspirin while the COX-2 selective drug etoricoxib and the non-opioid analgesic paracetamol, both drugs have no acidic structure, failed to activate AMPK. In conclusion, our results revealed that AMPK can be activated by specific non-steroidal anti-inflammatory drugs such as salicylic acid, ibuprofen or diclofenac possibly depending on the acidic structure of the drugs. AMPK might therefore contribute to their antinociceptive and anti-inflammatory properties. PMID:26049010

  3. Identification of a protein kinase activity in purified foot- and-mouth disease virus.

    OpenAIRE

    Grubman, M J; Baxt, B; La Torre, J L; Bachrach, H L

    1981-01-01

    Purified preparations of foot-and-mouth disease virus types A, O, and C contain a protein kinase activity which can transfer the gamma phosphate of [32P]ATP to virion structural proteins VP2 and VP3 and exogenous acceptor proteins. Utilizing protamine sulfate as an acceptor, the kinase activity can be demonstrated in disrupted virus but not in intact virus. The enzyme is heat labile with optimal activity at pH 7 or greater. Serine residues of protamine sulfate were identified as the amino aci...

  4. Implications of compound heterozygous insulin receptor mutations in congenital muscle fibre type disproportion myopathy for the receptor kinase activation

    DEFF Research Database (Denmark)

    Klein, H H; Müller, R; Vestergaard, H;

    1999-01-01

    We studied insulin receptor kinase activation in two brothers with congenital muscle fibre type disproportion myopathy and compound heterozygous mutations of the insulin receptor gene, their parents, and their unaffected brother. In the father who has a heterozygote Arg1174-->Gln mutation, in situ......% of the receptors to become insulin-dependently activated. The mother carries a point mutation at the last base pair in exon 17 which, due to abnormal alternative splicing, could lead to normally transcribed receptor or truncated receptor lacking the kinase region. Kinase activation was normal in the mother...... activation of the receptor kinase in skeletal muscle was reduced about 70%. Selection of only those receptors that bound to anti-phosphotyrosine antibody showed that these receptors had normal kinase activity and that the reduction in overall kinase activity was due to the inability of about 70...

  5. Activation of the cellular mitogen-activated protein kinase pathways ERK, P38 and JNK during Toxoplasma gondii invasion

    Directory of Open Access Journals (Sweden)

    Valère A.

    2003-03-01

    Full Text Available Host cell invasion is essential for the pathogenicity of the obligate intracellular protozoan parasite Toxoplasma gondii. In the present study, we evaluated the ability of T. gondii tachyzoites to trigger phosphorylation of the different mitogen-activated protein kinases (MAPK in human monocytic cells THP1. Kinetic experiments show that the peak of extracellular-signal-regulated kinase (ERK 1/2, P38 and cjun-NH2 terminal kinase (JNKs phosphorylation occurs between 10 and 60 min. The use of specific inhibitors of ERK1/2, P38 and JNK1/2 phosphorylation indicates the specificity of MAPKs phosphorylation during invasion. Signaling through cellular and parasite mitogen-activated protein (MAP kinase pathways appears to be critical for T. gondii invasion.

  6. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

    Science.gov (United States)

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-12-15

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  7. ALK kinase domain mutations in primary anaplastic large cell lymphoma: consequences on NPM-ALK activity and sensitivity to tyrosine kinase inhibitors.

    Directory of Open Access Journals (Sweden)

    Federica Lovisa

    Full Text Available ALK inhibitor crizotinib has shown potent antitumor activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL and the opportunity to include ALK inhibitors in first-line therapies is oncoming. However, recent studies suggest that crizotinib-resistance mutations may emerge in ALCL patients. In the present study, we analyzed ALK kinase domain mutational status of 36 paediatric ALCL patients at diagnosis to identify point mutations and gene aberrations that could impact on NPM-ALK gene expression, activity and sensitivity to small-molecule inhibitors. Amplicon ultra-deep sequencing of ALK kinase domain detected 2 single point mutations, R335Q and R291Q, in 2 cases, 2 common deletions of exon 23 and 25 in all the patients, and 7 splicing-related INDELs in a variable number of them. The functional impact of missense mutations and INDELs was evaluated. Point mutations were shown to affect protein kinase activity, signalling output and drug sensitivity. INDELs, instead, generated kinase-dead variants with dominant negative effect on NPM-ALK kinase, in virtue of their capacity of forming non-functional heterocomplexes. Consistently, when co-expressed, INDELs increased crizotinib inhibitory activity on NPM-ALK signal processing, as demonstrated by the significant reduction of STAT3 phosphorylation. Functional changes in ALK kinase activity induced by both point mutations and structural rearrangements were resolved by molecular modelling and dynamic simulation analysis, providing novel insights into ALK kinase domain folding and regulation. Therefore, these data suggest that NPM-ALK pre-therapeutic mutations may be found at low frequency in ALCL patients. These mutations occur randomly within the ALK kinase domain and affect protein activity, while preserving responsiveness to crizotinib.

  8. ALK kinase domain mutations in primary anaplastic large cell lymphoma: consequences on NPM-ALK activity and sensitivity to tyrosine kinase inhibitors.

    Science.gov (United States)

    Lovisa, Federica; Cozza, Giorgio; Cristiani, Andrea; Cuzzolin, Alberto; Albiero, Alessandro; Mussolin, Lara; Pillon, Marta; Moro, Stefano; Basso, Giuseppe; Rosolen, Angelo; Bonvini, Paolo

    2015-01-01

    ALK inhibitor crizotinib has shown potent antitumor activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL) and the opportunity to include ALK inhibitors in first-line therapies is oncoming. However, recent studies suggest that crizotinib-resistance mutations may emerge in ALCL patients. In the present study, we analyzed ALK kinase domain mutational status of 36 paediatric ALCL patients at diagnosis to identify point mutations and gene aberrations that could impact on NPM-ALK gene expression, activity and sensitivity to small-molecule inhibitors. Amplicon ultra-deep sequencing of ALK kinase domain detected 2 single point mutations, R335Q and R291Q, in 2 cases, 2 common deletions of exon 23 and 25 in all the patients, and 7 splicing-related INDELs in a variable number of them. The functional impact of missense mutations and INDELs was evaluated. Point mutations were shown to affect protein kinase activity, signalling output and drug sensitivity. INDELs, instead, generated kinase-dead variants with dominant negative effect on NPM-ALK kinase, in virtue of their capacity of forming non-functional heterocomplexes. Consistently, when co-expressed, INDELs increased crizotinib inhibitory activity on NPM-ALK signal processing, as demonstrated by the significant reduction of STAT3 phosphorylation. Functional changes in ALK kinase activity induced by both point mutations and structural rearrangements were resolved by molecular modelling and dynamic simulation analysis, providing novel insights into ALK kinase domain folding and regulation. Therefore, these data suggest that NPM-ALK pre-therapeutic mutations may be found at low frequency in ALCL patients. These mutations occur randomly within the ALK kinase domain and affect protein activity, while preserving responsiveness to crizotinib.

  9. Basal aurora kinase B activity is sufficient for histone H3 phosphorylation in prophase

    Directory of Open Access Journals (Sweden)

    Ly-Thuy-Tram Le

    2013-02-01

    Histone H3 phosphorylation is the hallmark of mitosis deposited by aurora kinase B. Benzo[e]pyridoindoles are a family of potent, broad, ATP-competitive aurora kinase inhibitors. However, benzo[e]pyridoindole C4 only inhibits histone H3 phosphorylation in prophase but not in metaphase. Under the C4 treatment, the cells enter into mitosis with dephosphorylated histone H3, assemble chromosomes normally and progress to metaphase, and then to anaphase. C4 also induces lagging chromosome in anaphase but we demonstrated that these chromosome compaction defects are not related to the absence of H3 phosphorylation in prophase. As a result of C4 action, mitosis lasts longer and the cell cycle is slowed down. We reproduced the mitotic defects with reduced concentrations of potent pan aurora kinase as well as with a specific aurora B ATP-competitive inhibitor; we therefore propose that histone H3 phosphorylation and anaphase chromosome compaction involve the basal activity of aurora kinase B. Our data suggest that aurora kinase B is progressively activated at mitosis entry and at anaphase onset. The full activation of aurora kinase B by its partners, in prometaphase, induces a shift in the catalytic domain of aurora B that modifies its affinity for ATP. These waves of activation/deactivation of aurora B correspond to different conformations of the chromosomal complex revealed by FRAP. The presence of lagging chromosomes may have deleterious consequences on the daughter cells and, unfortunately, the situation may be encountered in patients receiving treatment with aurora kinase inhibitors.

  10. Characterization of niphatenones that inhibit androgen receptor N-terminal domain.

    Directory of Open Access Journals (Sweden)

    Carmen A Banuelos

    Full Text Available Androgen ablation therapy causes a temporary reduction in tumor burden in patients with advanced prostate cancer. Unfortunately the malignancy will return to form lethal castration-recurrent prostate cancer (CRPC. The androgen receptor (AR remains transcriptionally active in CRPC in spite of castrate levels of androgens in the blood. AR transcriptional activity resides in its N-terminal domain (NTD. Possible mechanisms of continued AR transcriptional activity may include, at least in part, expression of constitutively active splice variants of AR that lack the C-terminal ligand-binding domain (LBD. Current therapies that target the AR LBD, would not be effective against these AR variants. Currently no drugs are clinically available that target the AR NTD which should be effective against these AR variants as well as full-length AR. Niphatenones were originally isolated and identified in active extracts from Niphates digitalis marine sponge. Here we begin to characterize the mechanism of niphatenones in blocking AR transcriptional activity. Both enantiomers had similar IC50 values of 6 µM for inhibiting the full-length AR in a functional transcriptional assay. However, (S-niphatenone had significantly better activity against the AR NTD compared to (R-niphatenone. Consistent with niphatenones binding to and inhibiting transactivation of AR NTD, niphatenones inhibited AR splice variant. Niphatenone did not affect the transcriptional activity of the related progesterone receptor, but slightly decreased glucocorticoid receptor (GR activity and covalently bound to GR activation function-1 (AF-1 region. Niphatenone blocked N/C interactions of AR without altering either AR protein levels or its intracellular localization in response to androgen. Alkylation with glutathione suggests that niphatenones are not a feasible scaffold for further drug development.

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

    DEFF Research Database (Denmark)

    Ard, Ryan; Mulatz, Kirk; Abramovici, Hanan;

    2012-01-01

    Rho GTPases share a common inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI), which regulates their expression levels, membrane localization, and activation state. The selective dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals, b...

  12. Antipeptide antibody that specifically inhibits insulin receptor autophosphorylation and protein kinase activity

    International Nuclear Information System (INIS)

    Two site-specific antibodies that immunoprecipitate the human insulin receptor have been prepared by immunizing rabbits with chemically synthesized peptides derived from the cDNA-predicted amino acid sequence of the β subunit of the proreceptor. Antibodies to the carboxyl terminus (AbP5) and to a domain around tyrosine-960 (AbP4) specifically recognize the β subunit of the receptor on immunoblots. Both antibodies immunoprecipitated 125I-labeled insulin-receptor complexes and the autophosphorylated receptor. Although neither antibody inhibited insulin binding to the receptor, both insulin-dependent autophosphorylation and exogenous substrate phosphorylation were inhibited by AbP4. Inhibition by AbP4 was dependent upon the phosphorylation state of the receptor; it was not detected when the receptor was autophosphorylated prior to addition of AbP4. AbP4 did not inhibit activity of the related epidermal growth factor (EGF)-receptor tyrosine protein kinase nor did it inhibit the activity of cAMP-dependent kinase or protein kinase C. The observation that an antibody directed to residues 952-967 of the proreceptor neutralizes the protein kinase activity of the β subunit suggest that this region may play a critical role in the function of the hormone-dependent, protein tyrosine-specific kinase activity of the insulin receptor

  13. Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase.

    Science.gov (United States)

    Clarke, Christopher J; Ohanian, Vasken; Ohanian, Jacqueline

    2007-05-01

    The phosphatidylinositol (PI) signaling pathway mediates norepinephrine (NE)- and endothelin-1 (ET-1)-stimulated vascular smooth muscle contraction through an inositol-trisphosphate-induced rise in intracellular calcium and diacylglycerol (DG) activation of protein kinase C (PKC). Subsequent activation of DG kinases (DGKs) metabolizes DG to phosphatidic acid (PA), potentially regulating PKC activity. Because precise regulation and spatial restriction of the PI pathway is necessary for specificity, we have investigated whether this occurs within caveolae/rafts, specialized plasma membrane microdomains implicated in vascular smooth muscle contraction. We show that components of the PI signaling cascade-phosphatidylinositol 4,5-bisphosphate (PIP(2)), PA, and DGK-theta are present in caveolae/rafts prepared from rat mesenteric small arteries. Stimulation with NE or ET-1 induced [(33)P]PIP(2) hydrolysis solely within caveolae/rafts. NE stimulated an increase in DGK activity in caveolae/rafts alone, whereas ET-1 activated DGK in caveolae/rafts and noncaveolae/rafts; however, [(33)P]PA increased in all fractions with both agonists. Previously, we reported that NE activated DGK-theta in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner; here, we describe PI3-kinase-dependent DGK activation and [(33)P]PA production in caveolae/rafts in response to NE but not ET-1. Additionally, PKB, a potential activator of DGK-theta, translocated to caveolae/rafts in response to NE but not ET-1, and PI3-kinase inhibition prevented this. Furthermore, PI3-kinase inhibition reduced the sensitivity of contraction to NE but not ET-1. Our study shows that caveolae/rafts are major sites of vasoconstrictor hormone activation of the PI pathway in intact small arteries and suggest a link between lipid signaling events within caveolae/rafts and contraction. PMID:17208990

  14. Uncoupling Intramolecular Processing and Substrate Hydrolysis in the N-terminal Nucleophile Hydrolase hASRGL1 by Circular Permutation

    OpenAIRE

    Li, Wenzong; Cantor, Jason R.; Yogesha, S.D.; Yang, Shirley; Chantranupong, Lynne; Liu, June Qingxia; Agnello, Giulia; Georgiou, George; Stone, Everett M.; Yan ZHANG

    2012-01-01

    The human asparaginase-like protein 1 (hASRGL1) catalyzes the hydrolysis of l-asparagine and isoaspartyl-dipeptides. As an N-terminal nucleophile (Ntn) hydrolase superfamily member, the active form of hASRGL1 is generated by an intramolecular cleavage step with Thr168 as the catalytic residue. However, in vitro, autoprocessing is incomplete (~50 %), fettering the biophysical characterization of hASRGL1. We circumvented this obstacle by constructing a circularly permuted hASRGL1 that uncoupled...

  15. Abelson tyrosine kinase links PDGFbeta receptor activation to cytoskeletal regulation of NMDA receptors in CA1 hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Beazely Michael A

    2008-12-01

    Full Text Available Abstract Background We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl, control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood. Results Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation. Conclusion This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.

  16. Diacylglycerol kinase theta and zeta isoforms : regulation of activity, protein binding partners and physiological functions

    NARCIS (Netherlands)

    Los, Alrik Pieter

    2007-01-01

    Diacylglycerol kinases (DGKs) phosphorylate the second messenger diacylglycerol (DAG) yielding phosphatidic acid (PA). In this thesis, we investigated which structural domains of DGKtheta are required for DGK activity. Furthermore, we showed that DGKzeta binds to and is activated by the Retinoblasto

  17. Fluorous-assisted metal chelate affinity extraction technique for analysis of protein kinase activity.

    Science.gov (United States)

    Hayama, Tadashi; Kiyokawa, Ena; Yoshida, Hideyuki; Imakyure, Osamu; Yamaguchi, Masatoshi; Nohta, Hitoshi

    2016-08-15

    We have developed a fluorous affinity-based extraction method for measurement of protein kinase activity. In this method, a fluorescent peptide substrate was phosphorylated by a protein kinase, and the obtained phosphopeptide was selectively captured with Fe(III)-immobilized perfluoroalkyliminodiacetic acid reagent via a metal chelate affinity technique. Next, the captured phosphopeptide was selectively extracted into a fluorous solvent mixture, tetradecafluorohexane and 1H,1H,2H,2H-tridecafluoro-1-n-octanol (3:1, v/v), using the specificity of fluorous affinity (fluorophilicity). In contrast, the remained substrate peptide in the aqueous (non-fluorous) phase was easily measured fluorimetrically. Finally, the enzyme activity could be assayed by measuring the decrease in fluorescence. The feasibility of this method was demonstrated by applying the method for measurement of the activity of cAMP-dependent protein kinase (PKA) using its substrate peptide (kemptide) pre-labeled with carboxytetramethylrhodamine (TAMRA).

  18. In vitro and in vivo assays of protein kinase CK2 activity.

    Science.gov (United States)

    Prudent, Renaud; Sautel, Céline F; Moucadel, Virginie; Laudet, Béatrice; Filhol, Odile; Cochet, Claude

    2010-01-01

    Protein kinase CK2 (formerly casein kinase 2) is recognized as a central component in the control of the cellular homeostasis; however, much remains unknown regarding its regulation and its implication in cellular transformation and carcinogenesis. Moreover, study of CK2 function and regulation in a cellular context is complicated by the dynamic multisubunit architecture of this protein kinase. Although a number of robust techniques are available to assay CK2 activity in vitro, there is a demand for sensitive and specific assays to evaluate its activity in living cells. We hereby provide a detailed description of several assays for monitoring the CK2 activity and its subunit interaction in living cells. The guidelines presented herein should enable researchers in the field to establish strategies for cellular screenings of CK2 inhibitors. PMID:21050938

  19. Polo kinase regulates the localization and activity of the chromosomal passenger complex in meiosis and mitosis in Drosophila melanogaster.

    Science.gov (United States)

    Carmena, Mar; Lombardia, Miguel Ortiz; Ogawa, Hiromi; Earnshaw, William C

    2014-11-01

    Cell cycle progression is regulated by members of the cyclin-dependent kinase (CDK), Polo and Aurora families of protein kinases. The levels of expression and localization of the key regulatory kinases are themselves subject to very tight control. There is increasing evidence that crosstalk between the mitotic kinases provides for an additional level of regulation. We have previously shown that Aurora B activates Polo kinase at the centromere in mitosis, and that the interaction between Polo and the chromosomal passenger complex (CPC) component INCENP is essential in this activation. In this report, we show that Polo kinase is required for the correct localization and activity of the CPC in meiosis and mitosis. Study of the phenotype of different polo allele combinations compared to the effect of chemical inhibition revealed significant differences in the localization and activity of the CPC in diploid tissues. Our results shed new light on the mechanisms that control the activity of Aurora B in meiosis and mitosis.

  20. Novel protein kinase signaling systems regulating lifespan identified by small molecule library screening using Drosophila.

    Directory of Open Access Journals (Sweden)

    Stephen R Spindler

    Full Text Available Protein kinase signaling cascades control most aspects of cellular function. The ATP binding domains of signaling protein kinases are the targets of most available inhibitors. These domains are highly conserved from mammals to flies. Herein we describe screening of a library of small molecule inhibitors of protein kinases for their ability to increase Drosophila lifespan. We developed an assay system which allowed screening using the small amounts of materials normally present in commercial chemical libraries. The studies identified 17 inhibitors, the majority of which targeted tyrosine kinases associated with the epidermal growth factor receptor (EGFR, platelet-derived growth factor (PDGF/vascular endothelial growth factor (VEGF receptors, G-protein coupled receptor (GPCR, Janus kinase (JAK/signal transducer and activator of transcription (STAT, the insulin and insulin-like growth factor (IGFI receptors. Comparison of the protein kinase signaling effects of the inhibitors in vitro defined a consensus intracellular signaling profile which included decreased signaling by p38MAPK (p38, c-Jun N-terminal kinase (JNK and protein kinase C (PKC. If confirmed, many of these kinases will be novel additions to the signaling cascades known to regulate metazoan longevity.

  1. Activation of a mitogen-activated protein kinase pathway in Arabidopsis by chitin.

    Science.gov (United States)

    Wan, Jinrong; Zhang, Shuqun; Stacey, Gary

    2004-03-01

    SUMMARY Chitin, a polysaccharide composed of beta-1-->4-linked N-acetyl-d-glucosamine, has been shown or implicated as a signal in plant defence and development. However, the key components of chitin perception and downstream signalling in non-leguminous plants are largely unknown. In recent years, mitogen-activated protein kinases (MAPKs) and their cascades were shown to transduce various extracellular stimuli into internal cellular responses. To investigate the possible involvement of MAPKs in chitin signalling in plants, the model plant Arabidopsis thaliana was treated with crab-shell chitin and also with the purified chitin oligomers (degree of polymerization, d.p. = 2-8). Both mRNA levels and kinase activity of two MAPK genes, AtMPK6 and AtMPK3, were monitored after treatment. The mRNA of AtMPK3 was strongly up-regulated by both chitin and its larger oligomers (d.p. = 6-8), but the mRNA of AtMPK6 did not appear to be regulated by these treatments. However, the kinase activity of both MAPKs was induced by chitin and the larger oligomers (d.p. = 6-8), with AtMPK6 much more strongly induced. In addition, WRKY22, WRKY29, WRKY33 and WRKY53, which encode four WRKY transcription factors that recognize TTGAC(C/T) W-box elements in promoters of numerous plant defence-related genes, were up-regulated by these treatments. WRKY33 and WRKY53 expression was induced by the transgenic expression of the tobacco MAPKK NtMEK2 active mutant NtMEK2(DD), suggesting a potential role for these WRKY transcription factors in relaying the signal generated from the MAPK cascade to downstream genes. These data suggest that AtMPK6/AtMPK3 and WRKY transcription factors (such as WRKY33 and WRKY53) may be important components of a pathway involved in chitin signalling in Arabidopsis plants.

  2. Biochemi