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Sample records for arg kinase regulates

  1. One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton

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    Bianchi, Cristina; Torsello, Barbara; Di Stefano, Vitalba; Zipeto, Maria A.; Facchetti, Rita; Bombelli, Silvia; Perego, Roberto A., E-mail: roberto.perego@unimib.it

    2013-08-01

    The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness. -Highlights: • Each of the eight Arg isoforms was transfected in COS-7 cells. • Only the 1BSCTS Arg isoform has a nuclear distribution in transfected cells. • The cytoplasmic isoforms and F-actin colocalize cortically and in cell protrusions. • Arg isoforms differently phosphorylate p190RhoGAP and CrkII. • Arg isoforms differently modulate stress fibers, cell protrusions and motility.

  2. Regulation of Autophagy by Kinases

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    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  3. Retinoic acid promotes the development of Arg1-expressing dendritic cells for the regulation of T-cell differentiation

    OpenAIRE

    Chang, Jinsam; Thangamani, Shankar; Kim, Myung H.; Ulrich, Benjamin; Morris, Sidney M.; Chang H Kim

    2013-01-01

    Arginase I (Arg1), an enzyme expressed by many cell types including myeloid cells, can regulate immune responses. Expression of Arg1 in myeloid cells is regulated by a number of cytokines and tissue factors that influence cell development and activation. Retinoic acid, produced from vitamin A, regulates the homing and differentiation of lymphocytes and plays important roles in the regulation of immunity and immune tolerance. We report here that optimal expression of Arg1 in dendritic cells re...

  4. Arg deficiency does not influence the course of Myelin Oligodendrocyte Glycoprotein (MOG35-55)-induced experimental autoimmune encephalomyelitis

    DEFF Research Database (Denmark)

    Jacobsen, Freja Aksel; Hulst, Camilla; Bäckström, Thomas;

    2016-01-01

    Background: Inhibition of Abl kinases has an ameliorating effect on the rodent model for multiple sclerosis, experimental autoimmune encephalomyelitis, and arrests lymphocyte activation. The family of Abl kinases consists of the Abl1/Abl and Abl2/Arg tyrosine kinases. While the Abl kinase has bee...... encephalomyelitis is not dependent on Arg, but Arg plays a role for the number of B cells in immunized mice. This might suggest a novel role for the Arg kinase in B-cell trafficking or regulation. Furthermore, the results suggest that Arg is important for normal embryonic development....

  5. Identification of a differentially expressed thymidine kinase gene related to tapping panel dryness syndrome in the rubber tree (Hevea brasiliensis Muell. Arg. by random amplified polymorphic DNA screening

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    Arjunan Thulaseedharan

    2010-01-01

    Full Text Available Tapping panel dryness (TPD syndrome is one of the latex yield affecting factors in the rubber tree (Hevea brasiliensis Mull. Arg.. Therefore, identification of a DNA marker will be highly useful for screening progenies in breeding programs. The major goal of this study was to detect genetic variations and/or identification of gene fragments among 37 Hevea clones by the random amplified polymorphic DNA “fingerprinting” technique. Different levels of DNA polymorphism were detected with various primers and a distinct polymorphic band (2.0 kb was obtained with OPA-17 primer. It was cloned into a plasmid vector for further sequence characterization and the nucleotide sequence shows homology with a novel putative plant thymidine kinase (TK gene, designated as HbTK (Hevea brasiliensis thymidine kinase; GenBank accession number AY130829. The protein HbTK has 67%, 65%, 64%, and 63% similarity to TK genes of Medicago, Oryza, Arabidopsis, and Lyco- persicon, respectively, and it was highly conserved in all species analyzed. The predicted amino acid sequence contained conserved domains of TK proteins in the C-terminal half. Southern blot analysis indicated that HbTK is one of the members of a small gene family. Northern blot results revealed that the expression of the HbTK gene was up-regulated in mature bark tissues of the healthy tree while it was down-regulated in the TPD-affected one. These results suggest that this gene may play important roles in maintaining active nucleotide metabolism during cell division at the tapped site of bark tissues in the healthy tree under stress (tapping conditions for normal latex biosynthesis.

  6. The mechanism of protein kinase C regulation

    Institute of Scientific and Technical Information of China (English)

    Julhash U. KAZI

    2011-01-01

    Protein kinase C (PKC) is a family ofserine/threonine protein kinases that plays a central role in transducing extracellular signals into a variety of intracellular responses ranging from cell proliferation to apoptosis.Nine PKC genes have been identified in the human genome,which encode 10 proteins.Each member of this protein kinase family displays distinct biochemical characteristics and is enriched in different cellular and subcellular locations.Activation of PKC has been implicated in the regulation of cell growth and differentiation.This review summarizes works of the past years in the field of PKC biochemistry that covers regulation and activation mechanism of different PKC isoforms.

  7. Regulation and function of TPL-2,an IκB kinase-regulated MAP kinase kinase kinase

    Institute of Scientific and Technical Information of China (English)

    Thorsten Gantke; Srividya Sriskantharajah; Steven C Ley

    2011-01-01

    The IκB kinase(IKK)complex plays a well-documented role in innate and adaptive immunity.This function has been widely attributed to its role as the central activator of the NF-κB family of transcription factors.However,another important consequence of IKK activation is the regulation of TPL-2,a MEK kinase that is required for activation of ERK-1/2 MAP kinases in myeioid cells following Toll-like receptor and TNF receptor stimulation.In unstimulated cells,TPL-2 is stoichiometrically complexed with the NF-κB inhibitory protein NF-κB1 p105,which blocks TPL-2 access to its substrate MEK,and the ubiquitin-binding protein ABIN-2(A20-binding inhibitor of NF-κB 2),both of which are required to maintain TPL-2 protein stability.Following agonist stimulation,the IKK complex phosphorylates p105,triggering its K48-1inked ubiquitination and degradation by the proteasome.This releases TPL-2 from p105-mediated inhibition,facilitating activation of MEK,in addition to modulating NF-κB activation by liberating associated Rel subunits for translocation into the nucleus.IKK-induced proteolysis of 0105,therefore,can directly regulate both NF-κB and ERK MAP kinase activation via NF-κB1 p105.TPL-2 is critical for production of the proinflammatory cytokine TNF during inflammatory responses.Consequently,there has been considerable interest in the pharmaceutical industry to develop selective TPL-2 inhibitors as drugs for the treatment of TNF-dependent inflammatory,diseases,such as rheumatoid arthritis and inflammatory bowel disease.This review summarizes our current understanding of the regulation of TPL-2 signaling function,and also the complex positive and negative roles of TPL-2 in immune and inflammatory responses.

  8. Abl family kinases regulate endothelial barrier function in vitro and in mice.

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    Elizabeth M Chislock

    Full Text Available The maintenance of endothelial barrier function is essential for normal physiology, and increased vascular permeability is a feature of a wide variety of pathological conditions, leading to complications including edema and tissue damage. Use of the pharmacological inhibitor imatinib, which targets the Abl family of non-receptor tyrosine kinases (Abl and Arg, as well as other tyrosine kinases including the platelet-derived growth factor receptor (PDGFR, Kit, colony stimulating factor 1 receptor (CSF1R, and discoidin domain receptors, has shown protective effects in animal models of inflammation, sepsis, and other pathologies characterized by enhanced vascular permeability. However, the imatinib targets involved in modulation of vascular permeability have not been well-characterized, as imatinib inhibits multiple tyrosine kinases not only in endothelial cells and pericytes but also immune cells important for disorders associated with pathological inflammation and abnormal vascular permeability. In this work we employ endothelial Abl knockout mice to show for the first time a direct role for Abl in the regulation of vascular permeability in vivo. Using both Abl/Arg-specific pharmacological inhibition and endothelial Abl knockout mice, we demonstrate a requirement for Abl kinase activity in the induction of endothelial permeability by vascular endothelial growth factor both in vitro and in vivo. Notably, Abl kinase inhibition also impaired endothelial permeability in response to the inflammatory mediators thrombin and histamine. Mechanistically, we show that loss of Abl kinase activity was accompanied by activation of the barrier-stabilizing GTPases Rac1 and Rap1, as well as inhibition of agonist-induced Ca(2+ mobilization and generation of acto-myosin contractility. In all, these findings suggest that pharmacological targeting of the Abl kinases may be capable of inhibiting endothelial permeability induced by a broad range of agonists and that use

  9. Selective regulation of MAP kinase signaling by an endomembrane phosphatidylinositol 4-kinase.

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    Cappell, Steven D; Dohlman, Henrik G

    2011-04-29

    Multiple MAP kinase pathways share components yet initiate distinct biological processes. Signaling fidelity can be maintained by scaffold proteins and restriction of signaling complexes to discreet subcellular locations. For example, the yeast MAP kinase scaffold Ste5 binds to phospholipids produced at the plasma membrane and promotes selective MAP kinase activation. Here we show that Pik1, a phosphatidylinositol 4-kinase that localizes primarily to the Golgi, also regulates MAP kinase specificity but does so independently of Ste5. Pik1 is required for full activation of the MAP kinases Fus3 and Hog1 and represses activation of Kss1. Further, we show by genetic epistasis analysis that Pik1 likely regulates Ste11 and Ste50, components shared by all three MAP kinase pathways, through their interaction with the scaffold protein Opy2. These findings reveal a new regulator of signaling specificity functioning at endomembranes rather than at the plasma membrane. PMID:21388955

  10. Cationic amino acid transporters and Salmonella Typhimurium ArgT collectively regulate arginine availability towards intracellular Salmonella growth.

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    Priyanka Das

    Full Text Available Cationic amino acid transporters (mCAT1 and mCAT2B regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells.

  11. Transcriptional Regulation of Pyruvate Dehydrogenase Kinase

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    Ji Yun Jeong

    2012-10-01

    Full Text Available The pyruvate dehydrogenase complex (PDC activity is crucial to maintains blood glucose and ATP levels, which largely depends on the phosphorylation status by pyruvate dehydrogenase kinase (PDK isoenzymes. Although it has been reported that PDC is phosphorylated and inactivated by PDK2 and PDK4 in metabolically active tissues including liver, skeletal muscle, heart, and kidney during starvation and diabetes, the precise mechanisms by which expression of PDK2 and PDK4 are transcriptionally regulated still remains unclear. Insulin represses the expression of PDK2 and PDK4 via phosphorylation of FOXO through PI3K/Akt signaling pathway. Several nuclear hormone receptors activated due to fasting or increased fat supply, including peroxisome proliferator-activated receptors, glucocorticoid receptors, estrogen-related receptors, and thyroid hormone receptors, also participate in the up-regulation of PDK2 and PDK4; however, the endogenous ligands that bind those nuclear receptors have not been identified. It has been recently suggested that growth hormone, adiponectin, epinephrine, and rosiglitazone also control the expression of PDK4 in tissue-specific manners. In this review, we discuss several factors involved in the expressional regulation of PDK2 and PDK4, and introduce current studies aimed at providing a better understanding of the molecular mechanisms that underlie the development of metabolic diseases such as diabetes.

  12. Transcriptional regulation of pyruvate dehydrogenase kinase.

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    Jeong, Ji Yun; Jeoung, Nam Ho; Park, Keun-Gyu; Lee, In-Kyu

    2012-10-01

    The pyruvate dehydrogenase complex (PDC) activity is crucial to maintains blood glucose and ATP levels, which largely depends on the phosphorylation status by pyruvate dehydrogenase kinase (PDK) isoenzymes. Although it has been reported that PDC is phosphorylated and inactivated by PDK2 and PDK4 in metabolically active tissues including liver, skeletal muscle, heart, and kidney during starvation and diabetes, the precise mechanisms by which expression of PDK2 and PDK4 are transcriptionally regulated still remains unclear. Insulin represses the expression of PDK2 and PDK4 via phosphorylation of FOXO through PI3K/Akt signaling pathway. Several nuclear hormone receptors activated due to fasting or increased fat supply, including peroxisome proliferator-activated receptors, glucocorticoid receptors, estrogen-related receptors, and thyroid hormone receptors, also participate in the up-regulation of PDK2 and PDK4; however, the endogenous ligands that bind those nuclear receptors have not been identified. It has been recently suggested that growth hormone, adiponectin, epinephrine, and rosiglitazone also control the expression of PDK4 in tissue-specific manners. In this review, we discuss several factors involved in the expressional regulation of PDK2 and PDK4, and introduce current studies aimed at providing a better understanding of the molecular mechanisms that underlie the development of metabolic diseases such as diabetes. PMID:23130316

  13. Kinase-Mediated Regulation of P4-ATPases

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    Frøsig, Merethe Mørch

    Abstract Kinase-Mediated Regulation of P4-ATPases Understanding kinase-mediated regulation and designing novel tools to study regulatory proteins of P4-ATPases P4-ATPases play a critical role in the biogenesis of transport vesicles in the secretory and endocytic pathways, and P4-ATPase activity...

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

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    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

  15. Regulation of tomato Prf by Pto-like protein kinases.

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    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.

  16. 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.

  17. 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.

  18. Regulation of the interaction between protein kinase C-related protein kinase 2 (PRK2) and its upstream kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1)

    DEFF Research Database (Denmark)

    Dettori, Rosalia; Sonzogni, Silvina; Meyer, Lucas;

    2009-01-01

    The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of...... numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by (32)P(i) revealed phosphorylation at two sites, the...... the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2...

  19. Mst1 and Mst2 kinases: regulations and diseases

    OpenAIRE

    Qin, Funiu; Tian, Jing; Zhou, Dawang; Chen, Lanfen

    2013-01-01

    The Hippo signaling pathway has emerged as a critical regulator for organ size control. The serine/threonine protein kinases Mst1 and Mst2, mammalian homologs of the Hippo kinase from Drosophila, play the central roles in the Hippo pathway controlling the cell proliferation, differentiation, and apoptosis during development. Mst1/2 can be activated by cellular stressors and the activation of Mst1/2 might enforce a feedback stimulation system to regulate oxidant levels through several mechanis...

  20. Transcriptional Regulation of Pyruvate Dehydrogenase Kinase

    OpenAIRE

    Ji Yun Jeong; Nam Ho Jeoung; Keun-Gyu Park; In-Kyu Lee

    2012-01-01

    The pyruvate dehydrogenase complex (PDC) activity is crucial to maintains blood glucose and ATP levels, which largely depends on the phosphorylation status by pyruvate dehydrogenase kinase (PDK) isoenzymes. Although it has been reported that PDC is phosphorylated and inactivated by PDK2 and PDK4 in metabolically active tissues including liver, skeletal muscle, heart, and kidney during starvation and diabetes, the precise mechanisms by which expression of PDK2 and PDK4 are transcriptionally re...

  1. 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

  2. Novel regulation of protein kinase C-η

    OpenAIRE

    Pal, Deepanwita; Outram, Shalini Persaud; Basu, Alakananda

    2012-01-01

    Protein kinase C (PKC) is the receptor for tumor promoting phorbol esters, which are potent activators of conventional and novel PKCs, but persistent treatment with phorbol esters leads to downregulation of these PKCs. However, PKCη, a novel PKC isozyme, resists downregulation by tumor-promoting phorbol esters, but little is known about how PKCη level is regulated. Phosphorylation and dephosphorylation play an important role in regulating activity and stability of PKCs. In the present study, ...

  3. Regulation of Multicellular Spheroids by MAPK and FYN Kinase.

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    Lee, Casey; Ramos, Daniel M

    2016-08-01

    Understanding of the biology of oral squamous cell carcinoma (SCC) has not progressed significantly in the past 60 years, with 5-year survival remaining at approximately 50%. The epidemic of Human Papilloma Virus and its associated SCC warrants a renewed emphasis on fully understanding this disease. We previously used the 3-dimensional multicellular spheroid (MCS) model system to evaluate SCC behavior more accurately. In this study, we determined that SCC growth in MCS approximates epithelial to mesenchymal transition. Organization of an MCS requires the full-length β6 integrin subunit and its maintenance requires mitogen-activated protein kinase (MAPK). Limiting FYN kinase activation results in the down-regulation of E-cadherin, β-catenin and an increase in expression of N-cadherin and SNAIL. These results indicate that the microenvironment and growth patterns in an MCS are complex and require MAPK and FYN kinase. PMID:27466485

  4. Regulation of polar auxin transport by protein and lipid kinases.

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    Armengot, Laia; Marquès-Bueno, Maria Mar; Jaillais, Yvon

    2016-07-01

    The directional transport of auxin, known as polar auxin transport (PAT), allows asymmetric distribution of this hormone in different cells and tissues. This system creates local auxin maxima, minima, and gradients that are instrumental in both organ initiation and shape determination. As such, PAT is crucial for all aspects of plant development but also for environmental interaction, notably in shaping plant architecture to its environment. Cell to cell auxin transport is mediated by a network of auxin carriers that are regulated at the transcriptional and post-translational levels. Here we review our current knowledge on some aspects of the 'non-genomic' regulation of auxin transport, placing an emphasis on how phosphorylation by protein and lipid kinases controls the polarity, intracellular trafficking, stability, and activity of auxin carriers. We describe the role of several AGC kinases, including PINOID, D6PK, and the blue light photoreceptor phot1, in phosphorylating auxin carriers from the PIN and ABCB families. We also highlight the function of some receptor-like kinases (RLKs) and two-component histidine kinase receptors in PAT, noting that there are probably RLKs involved in co-ordinating auxin distribution yet to be discovered. In addition, we describe the emerging role of phospholipid phosphorylation in polarity establishment and intracellular trafficking of PIN proteins. We outline these various phosphorylation mechanisms in the context of primary and lateral root development, leaf cell shape acquisition, as well as root gravitropism and shoot phototropism. PMID:27242371

  5. Cell fate regulation governed by a repurposed bacterial histidine kinase.

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    W Seth Childers

    2014-10-01

    Full Text Available One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK∼P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.

  6. 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

  7. Susi, a negative regulator of Drosophila PI3-kinase.

    Science.gov (United States)

    Wittwer, Franz; Jaquenoud, Malika; Brogiolo, Walter; Zarske, Marcel; Wüstemann, Philipp; Fernandez, Rafael; Stocker, Hugo; Wymann, Matthias P; Hafen, Ernst

    2005-06-01

    The Phosphatidylinositol-3 kinase/Protein Kinase B (PI3K/PKB) signaling pathway controls growth, metabolism, and lifespan in animals, and deregulation of its activity is associated with diabetes and cancer in humans. Here, we describe Susi, a coiled-coil domain protein that acts as a negative regulator of insulin signaling in Drosophila. Whereas loss of Susi function increases body size, overexpression of Susi reduces growth. We provide genetic evidence that Susi negatively regulates dPI3K activity. Susi directly binds to dP60, the regulatory subunit of dPI3K. Since Susi has no overt similarity to known inhibitors of PI3K/PKB signaling, it defines a novel mechanism by which this signaling cascade is kept in check. The fact that Susi is expressed in a circadian rhythm, with highest levels during the night, suggests that Susi attenuates insulin signaling during the fasting period.

  8. Enzymatic Regulation of Cytosolic Thymidine Kinase 1 and Mitochondrial Thymidine Kinase 2

    DEFF Research Database (Denmark)

    Munch-Petersen, Birgitte

    2010-01-01

    The central enzyme on the de novo pathway for synthesis of DNA precursors, the deoxyribonucleoside triphosphates, is ribonucleotide reductase (RNR). Deoxythymidine triphosphate (dTTP) has a key role in control of RNR activity shifting the specificity from pyrimidine to purine nucleotide reduction....... Apart from the complex de novo synthesis of dTTP through UDP reduction, dTTP is provided through salvage of thymidine catalyzed by the thymidine kinases, the cytosolic and cell cycle regulated TK1 and the mitochondrial and constitutively expressed TK2. The complex enzymatic regulation of TK1 and TK2 and...

  9. Scaffold Proteins Regulating Extracellular Regulated Kinase Function in Cardiac Hypertrophy and Disease

    OpenAIRE

    Liang, Yan; Sheikh, Farah

    2016-01-01

    The mitogen activated protein kinase (MAPK)-extracellular regulated kinase 1/2 (ERK1/2) pathway is a central downstream signaling pathway that is activated in cardiac muscle cells during mechanical and agonist-mediated hypertrophy. Studies in genetic mouse models deficient in ERK-associated MAPK components pathway have further reinforced a direct role for this pathway in stress-induced cardiac hypertrophy and disease. However, more recent studies have highlighted that these signaling pathways...

  10. Protein kinase C and extracellular signal-regulated kinase regulate movement, attachment, pairing and egg release in Schistosoma mansoni.

    Directory of Open Access Journals (Sweden)

    Margarida Ressurreição

    2014-06-01

    Full Text Available Protein kinases C (PKCs and extracellular signal-regulated kinases (ERKs are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using 'smart' antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance.

  11. Brassinosteroid regulated kinases (BRKs) that mediate brassinosteroid signal transduction and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhi-Yong; Tang, Wenqiang

    2013-09-24

    The present invention identifies a novel family of kinases regulated by brassinosteroids, referred to as BRKs (brassinosteroid regulated kinases) or BSKs (brassinosteroid signaling kinases). The present invention provides methods for modulating the response of a plant cell to a brassinosteroid using BRKs.

  12. MAP kinase phosphatase 2 regulates macrophage-adipocyte interaction.

    Directory of Open Access Journals (Sweden)

    Huipeng Jiao

    Full Text Available Inflammation is critical for the development of obesity-associated metabolic disorders. This study aims to investigate the role of mitogen-activated protein kinase phosphatase 2 (MKP-2 in inflammation during macrophage-adipocyte interaction.White adipose tissues (WAT from mice either on a high-fat diet (HFD or normal chow (NC were isolated to examine the expression of MKP-2. Murine macrophage cell line RAW264.7 stably expressing MKP-2 was used to study the regulation of MKP-2 in macrophages in response to saturated free fatty acid (FFA and its role in macrophage M1/M2 activation. Macrophage-adipocyte co-culture system was employed to investigate the role of MKP-2 in regulating inflammation during adipocyte-macrophage interaction. c-Jun N-terminal kinase (JNK- and p38-specific inhibitors were used to examine the mechanisms by which MKP-2 regulates macrophage activation and macrophage-adipocytes interaction.HFD changed the expression of MKP-2 in WAT, and MKP-2 was highly expressed in the stromal vascular cells (SVCs. MKP-2 inhibited the production of proinflammatory cytokines in response to FFA stimulation in macrophages. MKP-2 inhibited macrophage M1 activation through JNK and p38. In addition, overexpression of MKP-2 in macrophages suppressed inflammation during macrophage-adipocyte interaction.MKP-2 is a negative regulator of macrophage M1 activation through JNK and p38 and inhibits inflammation during macrophage-adipocyte interaction.

  13. Integrin-linked kinase regulates interphase and mitotic microtubule dynamics.

    Directory of Open Access Journals (Sweden)

    Simin Lim

    Full Text Available Integrin-linked kinase (ILK localizes to both focal adhesions and centrosomes in distinct multiprotein complexes. Its dual function as a kinase and scaffolding protein has been well characterized at focal adhesions, where it regulates integrin-mediated cell adhesion, spreading, migration and signaling. At the centrosomes, ILK regulates mitotic spindle organization and centrosome clustering. Our previous study showed various spindle defects after ILK knockdown or inhibition that suggested alteration in microtubule dynamics. Since ILK expression is frequently elevated in many cancer types, we investigated the effects of ILK overexpression on microtubule dynamics. We show here that overexpressing ILK in HeLa cells was associated with a shorter duration of mitosis and decreased sensitivity to paclitaxel, a chemotherapeutic agent that suppresses microtubule dynamics. Measurement of interphase microtubule dynamics revealed that ILK overexpression favored microtubule depolymerization, suggesting that microtubule destabilization could be the mechanism behind the decreased sensitivity to paclitaxel, which is known to stabilize microtubules. Conversely, the use of a small molecule inhibitor selective against ILK, QLT-0267, resulted in suppressed microtubule dynamics, demonstrating a new mechanism of action for this compound. We further show that treatment of HeLa cells with QLT-0267 resulted in higher inter-centromere tension in aligned chromosomes during mitosis, slower microtubule regrowth after cold depolymerization and the presence of a more stable population of spindle microtubules. These results demonstrate that ILK regulates microtubule dynamics in both interphase and mitotic cells.

  14. Creatine kinase in cell cycle regulation and cancer.

    Science.gov (United States)

    Yan, Yong-Bin

    2016-08-01

    The phosphocreatine-creatine kinase (CK) shuttle system is increasingly recognized as a fundamental mechanism for ATP homeostasis in both excitable and non-excitable cells. Many intracellular processes are ATP dependent. Cell division is a process requiring a rapid rate of energy turnover. Cell cycle regulation is also a key point to understanding the mechanisms underlying cancer progression. It has been known for about 40 years that aberrant CK levels are associated with various cancers and for over 30 years that CK is involved in mitosis regulation. However, the underlying molecular mechanisms have not been investigated sufficiently until recently. By maintaining ATP at sites of high-energy demand, CK can regulate cell cycle progression by affecting the intracellular energy status as well as by influencing signaling pathways that are essential to activate cell division and cytoskeleton reorganization. Aberrant CK levels may impair cell viability under normal or stressed conditions and induce cell death. The involvement of CK in cell cycle regulation and cellular energy metabolism makes it a potential diagnostic biomarker and therapeutic target in cancer. To understand the multiple physiological/pathological functions of CK, it is necessary to identify CK-binding partners and regulators including proteins, non-coding RNAs and participating endogenous small molecular weight chemical compounds. This review will focus on molecular mechanisms of CK in cell cycle regulation and cancer progression. It will also discuss the implications of recent mechanistic studies, the emerging problems and future challenges of the multifunctional enzyme CK. PMID:27020776

  15. 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

  16. Tricornered Kinase Regulates Synapse Development by Regulating the Levels of Wiskott-Aldrich Syndrome Protein.

    Directory of Open Access Journals (Sweden)

    Rajalaxmi Natarajan

    Full Text Available Precise regulation of synapses during development is essential to ensure accurate neural connectivity and function of nervous system. Many signaling pathways, including the mTOR (mechanical Target of Rapamycin pathway operate in neurons to maintain genetically determined number of synapses during development. mTOR, a kinase, is shared between two functionally distinct multi-protein complexes- mTORC1 and mTORC2, that act downstream of Tuberous Sclerosis Complex (TSC. We and others have suggested an important role for TSC in synapse development at the Drosophila neuromuscular junction (NMJ synapses. In addition, our data suggested that the regulation of the NMJ synapse numbers in Drosophila largely depends on signaling via mTORC2. In the present study, we further this observation by identifying Tricornered (Trc kinase, a serine/threonine kinase as a likely mediator of TSC signaling. trc genetically interacts with Tsc2 to regulate the number of synapses. In addition, Tsc2 and trc mutants exhibit a dramatic reduction in synaptic levels of WASP, an important regulator of actin polymerization. We show that Trc regulates the WASP levels largely, by regulating the transcription of WASP. Finally, we show that overexpression of WASP (Wiskott-Aldrich Syndrome Protein in trc mutants can suppress the increase in the number of synapses observed in trc mutants, suggesting that WASP regulates synapses downstream of Trc. Thus, our data provide a novel insight into how Trc may regulate the genetic program that controls the number of synapses during development.

  17. 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...

  18. Protein kinase D regulates cell death pathways in experimental pancreatitis

    Directory of Open Access Journals (Sweden)

    Jingzhen eYuan

    2012-03-01

    Full Text Available Inflammation and acinar cell necrosis are two major pathological responses of acute pancreatitis, a serious disorder with no current therapies directed to its molecular pathogenesis. Serine/threonine protein kinase D family, which includes PKD/PKD1, PKD2, and PKD3, has been increasingly implicated in the regulation of multiple physiological and pathophysiological effects. We recently reported that PKD/PKD1, the predominant PKD isoform expressed in rat pancreatic acinar cells, mediates early events of pancreatitis including NF-kappaB activation and inappropriate intracellular digestive enzyme activation. In current studies, we investigated the role and mechanisms of PKD/PKD1 in the regulation of necrosis in pancreatic acinar cells by using two novel small molecule PKD inhibitors CID755673 and CRT0066101 and molecular approaches in in vitro and in vivo experimental models of acute pancreatitis. Our results demonstrated that both CID755673 and CRT0066101 are PKD-specific inhibitors and that PKD/PKD1 inhibition by either the chemical inhibitors or specific PKD/PKD1 siRNAs attenuated necrosis while promoting apoptosis induced by pathological doses of cholecystokinin-octapeptide (CCK in pancreatic acinar cells. Conversely, upregulation of PKD expression in pancreatic acinar cells increased necrosis and decreased apoptosis. We further showed that PKD/PKD1 regulated several key cell death signals including inhibitors of apoptotic proteins (IAPs, caspases, receptor-interacting protein kinase 1 (RIP1 to promote necrosis. PKD/PKD1 inhibition by CID755673 significantly ameliorated necrosis and severity of pancreatitis in an in vivo experimental model of acute pancreatitis. Thus, our studies indicate that PKD/PKD1 is a key mediator of necrosis in acute pancreatitis and that PKD/PKD1 may represent a potential therapeutic target in acute pancreatitis.

  19. Regulation of the MAPK pathway by raf kinase inhibitory protein.

    Science.gov (United States)

    Vandamme, Drieke; Herrero, Ana; Al-Mulla, Fahd; Kolch, Walter

    2014-01-01

    The Raf kinase inhibitor protein 1 (RKIP-1) was the first reported endogenous inhibitor of Raf-1-MEK-ERK/MAPK cascade, by interfering with the phosphorylation of MEK by Raf-1. However, RKIP's functions related to the MAPK signaling are far more complex. Newer data indicate that by modulating different protein-protein interactions, RKIP is involved in fine-tuning cell signaling, modulating ERK dynamics, and regulating cross talk between different pathways. Here, we describe the molecular mechanisms by which RKIP controls MAPK signaling at different levels and vice versa and its regulation via feedback phosphorylation. We also focus on several discrepancies and questions that remain, such as the RKIP binding regulation by Raf-1 N-region phosphorylation, the possible B-Raf inhibition, and the effects of RKIP-lipid binding. We also describe how RKIP's role as key signaling modulator of many cell fate decisions leads to the fact that fine control of RKIP activity and regulation is crucial to avoid pathological processes, such as metastasis, pulmonary arterial hypertension, and heart failure.

  20. Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction

    DEFF Research Database (Denmark)

    Frödin, M; Gammeltoft, S

    1999-01-01

    by growth factors, peptide hormones and neurotransmitters, and Jun kinase (JNK) and p38 MAPK, which are activated by cellular stress stimulus as well as growth factors. This review describes the family of 90 kDa ribosomal S6 kinases (RSK; also known as p90rsk or MAPK-activated protein kinase-1, MAPKAP-K1......-related kinases that are activated by ERK as well as p38 MAPK were discovered and named mitogen- and stress-activated protein kinases (MSK). A number of cellular functions of RSK have been proposed. (1) Regulation of gene expression via association and phosphorylation of transcriptional regulators including c-Fos...

  1. DMPD: Regulation of phagocyte migration and recruitment by Src-family kinases. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18385944 Regulation of phagocyte migration and recruitment by Src-family kinases. B...how Regulation of phagocyte migration and recruitment by Src-family kinases. PubmedID 18385944 Title Regulation of phagocyte migratio

  2. Detailed search for protein kinase(s) involved in plasma membrane H+-ATPase activity regulation of yeast cells.

    Science.gov (United States)

    Pereira, Renata R; Castanheira, Diogo; Teixeira, Janaina A; Bouillet, Leoneide E M; Ribeiro, Erica M C; Trópia, Maria M J; Alvarez, Florencia; Correa, Lygia F M; Mota, Bruno E F; Conceição, Luis Eduardo F R; Castro, Ieso M; Brandão, Rogelio L

    2015-03-01

    This study displays a screening using yeast strains deficient in protein kinases known to exist in Saccharomyces cerevisiae. From 95 viable single mutants, 20 mutants appear to be affected in the glucose-induced extracellular acidification. The mutants that are unaffected in calcium signaling were tested for their sensitivity to hygromycin B. Furthermore, we verified whether the remaining mutants produced enzymes that are appropriately incorporated at plasma membrane. Finally, we measure the kinetic properties of the enzyme in purified plasma membranes from glucose-starved as well as glucose-fermenting cells. We confirmed the kinase Ptk2 involvement in H(+)-ATPase regulation (increase of affinity for ATP). However, the identification of the kinase(s) responsible for phosphorylation that leads to an increase in Vmax appears to be more complex. Complementary experiments were performed to check how those protein kinases could be related to the control of the plasma membrane H(+)-ATPase and/or the potential membrane. In summary, our results did not permit us to identify the protein kinase(s) involved in regulating the catalytic efficiency of the plasma membrane H(+)-ATPase. Therefore, our results indicate that the current regulatory model based on the phosphorylation of two different sites located in the C-terminus tail of the enzyme could be inappropriate.

  3. Rho kinase acts as a downstream molecule to participate in protein kinaseregulation of vascular reactivity after hemorrhagic shock in rats.

    Science.gov (United States)

    Li, Tao; Zhu, Yu; Zang, Jia-tao; Peng, Xiao-yong; Lan, Dan; Yang, Guang-ming; Xu, Jing; Liu, Liang-ming

    2014-09-01

    Our previous study demonstrated that Rho kinase and protein kinase C (PKC) played important parts in the regulation of vascular reactivity after shock. Using superior mesenteric arteries (SMAs) from hemorrhagic shock rats and hypoxia-treated vascular smooth muscle cells (VSMCs), relationship of PKCε regulation of vascular reactivity to Rho kinase, as well as the signal transduction after shock, was investigated. The results showed that inhibition of Rho kinase with the Rho kinase-specific inhibitor Y-27632 antagonized the PKCε-specific agonist carbachol and highly expressed PKCε-induced increase of vascular reactivity in SMAs and VSMCs, whereas inhibition of PKCε with its specific inhibitory peptide did not antagonize the Rho kinase agonist (U-46619)-induced increase of vascular reactivity in SMAs and VSMCs. Activation of PKCε or highly expressed PKCε upregulated the activity of Rho kinase and the phosphorylation of PKC-dependent phosphatase inhibitor 17 (CPI-17), zipper interacting protein kinase (ZIPK), and integrin-linked kinase (ILK), whereas activation of Rho kinase increased only CPI-17 phosphorylation. The specific neutralization antibodies of ZIPK and ILK antagonized PKCε-induced increases in the activity of Rho kinase, but CPI-17 neutralization antibody did not antagonize this effect. These results suggested that Rho kinase takes part in the regulation of PKCε on vascular reactivity after shock. Rho kinase is downstream of PKCε. Protein kinase Cε activates Rho kinase via ZIPK and ILK; CPI-17 is downstream of Rho kinase.

  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. Receptor Tyrosine Kinases: Molecular Switches Regulating CNS Axon Regeneration

    Directory of Open Access Journals (Sweden)

    Vasanthy Vigneswara

    2012-01-01

    Full Text Available The poor or lack of injured adult central nervous system (CNS axon regeneration results in devastating consequences and poor functional recovery. The interplay between the intrinsic and extrinsic factors contributes to robust inhibition of axon regeneration of injured CNS neurons. The insufficient or lack of trophic support for injured neurons is considered as one of the major obstacles contributing to their failure to survive and regrow their axons after injury. In the CNS, many of the signalling pathways associated with neuronal survival and axon regeneration are regulated by several classes of receptor tyrosine kinases (RTK that respond to a variety of ligands. This paper highlights and summarises the most relevant recent findings pertinent to different classes of the RTK family of molecules, with a particular focus on elucidating their role in CNS axon regeneration.

  6. Inositol Hexakisphosphate Kinase 3 Regulates Metabolism and Lifespan in Mice.

    Science.gov (United States)

    Moritoh, Yusuke; Oka, Masahiro; Yasuhara, Yoshitaka; Hozumi, Hiroyuki; Iwachidow, Kimihiko; Fuse, Hiromitsu; Tozawa, Ryuichi

    2016-01-01

    Inositol hexakisphosphate kinase 3 (IP6K3) generates inositol pyrophosphates, which regulate diverse cellular functions. However, little is known about its own physiological role. Here, we show the roles of IP6K3 in metabolic regulation. We detected high levels of both mouse and human IP6K3 mRNA in myotubes and muscle tissues. In human myotubes, IP6K3 was upregulated by dexamethasone treatment, which is known to inhibit glucose metabolism. Furthermore, Ip6k3 expression was elevated under diabetic, fasting, and disuse conditions in mouse skeletal muscles. Ip6k3(-/-) mice demonstrated lower blood glucose, reduced circulating insulin, deceased fat mass, lower body weight, increased plasma lactate, enhanced glucose tolerance, lower glucose during an insulin tolerance test, and reduced muscle Pdk4 expression under normal diet conditions. Notably, Ip6k3 deletion extended animal lifespan with concomitant reduced phosphorylation of S6 ribosomal protein in the heart. In contrast, Ip6k3(-/-) mice showed unchanged skeletal muscle mass and no resistance to the effects of high fat diet. The current observations suggest novel roles of IP6K3 in cellular regulation, which impact metabolic control and lifespan. PMID:27577108

  7. Inositol Hexakisphosphate Kinase 3 Regulates Metabolism and Lifespan in Mice

    Science.gov (United States)

    Moritoh, Yusuke; Oka, Masahiro; Yasuhara, Yoshitaka; Hozumi, Hiroyuki; Iwachidow, Kimihiko; Fuse, Hiromitsu; Tozawa, Ryuichi

    2016-01-01

    Inositol hexakisphosphate kinase 3 (IP6K3) generates inositol pyrophosphates, which regulate diverse cellular functions. However, little is known about its own physiological role. Here, we show the roles of IP6K3 in metabolic regulation. We detected high levels of both mouse and human IP6K3 mRNA in myotubes and muscle tissues. In human myotubes, IP6K3 was upregulated by dexamethasone treatment, which is known to inhibit glucose metabolism. Furthermore, Ip6k3 expression was elevated under diabetic, fasting, and disuse conditions in mouse skeletal muscles. Ip6k3−/− mice demonstrated lower blood glucose, reduced circulating insulin, deceased fat mass, lower body weight, increased plasma lactate, enhanced glucose tolerance, lower glucose during an insulin tolerance test, and reduced muscle Pdk4 expression under normal diet conditions. Notably, Ip6k3 deletion extended animal lifespan with concomitant reduced phosphorylation of S6 ribosomal protein in the heart. In contrast, Ip6k3−/− mice showed unchanged skeletal muscle mass and no resistance to the effects of high fat diet. The current observations suggest novel roles of IP6K3 in cellular regulation, which impact metabolic control and lifespan. PMID:27577108

  8. Regulation of the Saccharomyces cerevisiae EKI1-encoded Ethanolamine Kinase by Zinc Depletion*

    OpenAIRE

    Kersting, Michael C.; Carman, George M.

    2006-01-01

    Ethanolamine kinase catalyzes the committed step in the synthesis of phosphatidylethanolamine via the CDP-ethanolamine branch of the Kennedy pathway. Regulation of the EKI1-encoded ethanolamine kinase by the essential nutrient zinc was examined in Saccharomyces cerevisiae. The level of ethanolamine kinase activity increased when zinc was depleted from the growth medium. This regulation correlated with increases in the CDP-ethanolamine pathway intermediates phosphoethanolamine and CDP-ethanola...

  9. Protein kinase A regulates molecular chaperone transcription and protein aggregation.

    Directory of Open Access Journals (Sweden)

    Yue Zhang

    Full Text Available Heat shock factor 1 (HSF1 regulates one of the major pathways of protein quality control and is essential for deterrence of protein-folding disorders, particularly in neuronal cells. However, HSF1 activity declines with age, a change that may open the door to progression of neurodegenerative disorders such as Huntington's disease. We have investigated mechanisms of HSF1 regulation that may become compromised with age. HSF1 binds stably to the catalytic domain of protein kinase A (PKAcα and becomes phosphorylated on at least one regulatory serine residue (S320. We show here that PKA is essential for effective transcription of HSP genes by HSF1. PKA triggers a cascade involving HSF1 binding to the histone acetylase p300 and positive translation elongation factor 1 (p-TEFb and phosphorylation of the c-terminal domain of RNA polymerase II, a key mechanism in the downstream steps of HSF1-mediated transcription. This cascade appears to play a key role in protein quality control in neuronal cells expressing aggregation-prone proteins with long poly-glutamine (poly-Q tracts. Such proteins formed inclusion bodies that could be resolved by HSF1 activation during heat shock. Resolution of the inclusions was inhibited by knockdown of HSF1, PKAcα, or the pTEFb component CDK9, indicating a key role for the HSF1-PKA cascade in protein quality control.

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

    Science.gov (United States)

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

    2010-01-01

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

  11. Palmitylation of Src family tyrosine kinases regulates functional interaction with a B cell substrate.

    Science.gov (United States)

    Saouaf, S J; Wolven, A; Resh, M D; Bolen, J B

    1997-05-19

    Palmitylation of Src family tyrosine kinases has been shown to play a role in directing their membrane localization. Here we demonstrate that palmitylation can also regulate recognition and tyrosine phosphorylation of the B cell Src kinase substrate Ig alpha. Blk and Src, which are not palmitylated, phosphorylate co-expressed Ig alpha in Cos cells, whereas palmitylated Src kinases do not. Addition of a palmitylation site to Blk abrogates its phosphorylation of the substrate, while mutation of Fyn's palmitylation sites results in recognition and phosphorylation of Ig alpha. These results indicate that palmitylation, a reversible protein modification, aids in regulating recognition of physiologic substrates by Src family tyrosine kinases. PMID:9177269

  12. Pre-LTP requires extracellular signal-regulated kinase in the ACC

    Science.gov (United States)

    Yamanaka, Manabu; Tian, Zhen; Darvish-Ghane, Soroush

    2016-01-01

    The extracellular signal-regulated kinase is an important protein kinase for cortical plasticity. Long-term potentiation in the anterior cingulate cortex is believed to play important roles in chronic pain, fear, and anxiety. Previous studies of extracellular signal-regulated kinase are mainly focused on postsynaptic form of long-term potentiation (post-long-term potentiation). Little is known about the relationship between extracellular signal-regulated kinase and presynaptic long-term potentiation (pre-long-term potentiation) in cortical synapses. In this study, we examined whether pre-long-term potentiation in the anterior cingulate cortex requires the activation of presynaptic extracellular signal-regulated kinase. We found that p42/p44 mitogen-activated protein kinase inhibitors, PD98059 and U0126, suppressed the induction of pre-long-term potentiation. By contrast, these inhibitors did not affect the maintenance of pre-long-term potentiation. Using pharmacological inhibitors, we found that pre-long-term potentiation recorded for 1 h did not require transcriptional or translational processes. Our results strongly indicate that the activation of presynaptic extracellular signal-regulated kinase is required for the induction of pre-long-term potentiation, and this involvement may explain the contribution of extracellular signal-regulated kinase to mood disorders. PMID:27178245

  13. 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

  14. 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.

  15. 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

  16. PHYTOCHROME KINASE SUBSTRATE1 regulates root phototropism and gravitropism.

    Science.gov (United States)

    Boccalandro, Hernán E; De Simone, Silvia N; Bergmann-Honsberger, Ariane; Schepens, Isabelle; Fankhauser, Christian; Casal, Jorge J

    2008-01-01

    Light promotes the expression of PHYTOCHROME KINASE SUBSTRATE1 (PKS1) in the root of Arabidopsis thaliana, but the function of PKS1 in this organ is unknown. Unilateral blue light induced a negative root phototropic response mediated by phototropin 1 in wild-type seedlings. This response was absent in pks1 mutants. In the wild type, unilateral blue light enhanced PKS1 expression in the subapical region of the root several hours before bending was detectable. The negative phototropism and the enhanced PKS1 expression in response to blue light required phytochrome A (phyA). In addition, the pks1 mutation enhanced the root gravitropic response when vertically oriented seedlings were placed horizontally. The negative regulation of gravitropism by PKS1 occurred even in dark-grown seedlings and did not require phyA. Blue light also failed to induce negative phototropism in pks1 under reduced gravitational stimulation, indicating that the effect of pks1 on phototropism is not simply the consequence of the counteracting effect of enhanced gravitropism. We propose a model where the background level of PKS1 reduces gravitropism. After a phyA-dependent increase in its expression, PKS1 positively affects root phototropism and both effects contribute to negative curvature in response to unilateral blue light.

  17. Transcriptional regulation by protein kinase A in Cryptococcus neoformans.

    Directory of Open Access Journals (Sweden)

    Guanggan Hu

    2007-03-01

    Full Text Available A defect in the PKA1 gene encoding the catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP-dependent protein kinase A (PKA is known to reduce capsule size and attenuate virulence in the fungal pathogen Cryptococcus neoformans. Conversely, loss of the PKA regulatory subunit encoded by pkr1 results in overproduction of capsule and hypervirulence. We compared the transcriptomes between the pka1 and pkr1 mutants and a wild-type strain, and found that PKA influences transcript levels for genes involved in cell wall synthesis, transport functions such as iron uptake, the tricarboxylic acid cycle, and glycolysis. Among the myriad of transcriptional changes in the mutants, we also identified differential expression of ribosomal protein genes, genes encoding stress and chaperone functions, and genes for secretory pathway components and phospholipid synthesis. The transcriptional influence of PKA on these functions was reminiscent of the linkage between transcription, endoplasmic reticulum stress, and the unfolded protein response in Saccharomyces cerevisiae. Functional analyses confirmed that the PKA mutants have a differential response to temperature stress, caffeine, and lithium, and that secretion inhibitors block capsule production. Importantly, we also found that lithium treatment limits capsule size, thus reinforcing potential connections between this virulence trait and inositol and phospholipid metabolism. In addition, deletion of a PKA-regulated gene, OVA1, revealed an epistatic relationship with pka1 in the control of capsule size and melanin formation. OVA1 encodes a putative phosphatidylethanolamine-binding protein that appears to negatively influence capsule production and melanin accumulation. Overall, these findings support a role for PKA in regulating the delivery of virulence factors such as the capsular polysaccharide to the cell surface and serve to highlight the importance of secretion and phospholipid metabolism as potential

  18. Extracellular signal-regulated kinase-2 within the ventral tegmental area regulates responses to stress.

    Science.gov (United States)

    Iñiguez, Sergio D; Vialou, Vincent; Warren, Brandon L; Cao, Jun-Li; Alcantara, Lyonna F; Davis, Lindsey C; Manojlovic, Zarko; Neve, Rachael L; Russo, Scott J; Han, Ming-Hu; Nestler, Eric J; Bolaños-Guzmán, Carlos A

    2010-06-01

    Neurotrophic factors and their signaling pathways have been implicated in the neurobiological adaptations in response to stress and the regulation of mood-related behaviors. A candidate signaling molecule implicated in mediating these cellular responses is the extracellular signal-regulated kinase (ERK1/2), although its functional role in mood regulation remains to be fully elucidated. Here we show that acute (1 d) or chronic (4 weeks) exposure to unpredictable stress increases phosphorylation of ERK1/2 and of two downstream targets (ribosomal S6 kinase and mitogen- and stress-activated protein kinase 1) within the ventral tegmental area (VTA), an important substrate for motivated behavior and mood regulation. Using herpes simplex virus-mediated gene transfer to assess the functional significance of this ERK induction, we show that overexpressing ERK2 within the VTA increases susceptibility to stress as measured in the forced swim test, responses to unconditioned nociceptive stimuli, and elevated plus maze in Sprague Dawley male rats, and in the tail suspension test and chronic social defeat stress procedure in C57BL/6 male mice. In contrast, blocking ERK2 activity in the VTA produces stress-resistant behavioral responses in these same assays and also blocks a chronic stress-induced reduction in sucrose preference. The effects induced by ERK2 blockade were accompanied by decreases in the firing frequency of VTA dopamine neurons, an important electrophysiological hallmark of resilient-like behavior. Together, these results strongly implicate a role for ERK2 signaling in the VTA as a key modulator of responsiveness to stress and mood-related behaviors. PMID:20519540

  19. 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

  20. Co-conserved features associated with cis regulation of ErbB tyrosine kinases.

    Directory of Open Access Journals (Sweden)

    Amar Mirza

    Full Text Available BACKGROUND: The epidermal growth factor receptor kinases, or ErbB kinases, belong to a large sub-group of receptor tyrosine kinases (RTKs, which share a conserved catalytic core. The catalytic core of ErbB kinases have functionally diverged from other RTKs in that they are activated by a unique allosteric mechanism that involves specific interactions between the kinase core and the flanking Juxtamembrane (JM and COOH-terminal tail (C-terminal tail. Although extensive studies on ErbB and related tyrosine kinases have provided important insights into the structural basis for ErbB kinase functional divergence, the sequence features that contribute to the unique regulation of ErbB kinases have not been systematically explored. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we use a Bayesian approach to identify the selective sequence constraints that most distinguish ErbB kinases from other receptor tyrosine kinases. We find that strong ErbB kinase-specific constraints are imposed on residues that tether the JM and C-terminal tail to key functional regions of the kinase core. A conserved RIxKExE motif in the JM-kinase linker region and a glutamine in the inter-lobe linker are identified as two of the most distinguishing features of the ErbB family. While the RIxKExE motif tethers the C-terminal tail to the N-lobe of the kinase domain, the glutamine tethers the C-terminal tail to hinge regions critical for inter-lobe movement. Comparison of the active and inactive crystal structures of ErbB kinases indicates that the identified residues are conformationally malleable and can potentially contribute to the cis regulation of the kinase core by the JM and C-terminal tail. ErbB3, and EGFR orthologs in sponges and parasitic worms, diverge from some of the canonical ErbB features, providing insights into sub-family and lineage-specific functional specialization. CONCLUSION/SIGNIFICANCE: Our analysis pinpoints key residues for mutational analysis, and

  1. Recent Progress on Liver Kinase B1 (LKB1: Expression, Regulation, Downstream Signaling and Cancer Suppressive Function

    Directory of Open Access Journals (Sweden)

    Ren-You Gan

    2014-09-01

    Full Text Available Liver kinase B1 (LKB1, known as a serine/threonine kinase, has been identified as a critical cancer suppressor in many cancer cells. It is a master upstream kinase of 13 AMP-activated protein kinase (AMPK-related protein kinases, and possesses versatile biological functions. LKB1 gene is mutated in many cancers, and its protein can form different protein complexes with different cellular localizations in various cell types. The expression of LKB1 can be regulated through epigenetic modification, transcriptional regulation and post-translational modification. LKB1 dowcnstream pathways mainly include AMPK, microtubule affinity regulating kinase (MARK, salt-inducible kinase (SIK, sucrose non-fermenting protein-related kinase (SNRK and brain selective kinase (BRSK signalings, etc. This review, therefore, mainly discusses recent studies about the expression, regulation, downstream signaling and cancer suppressive function of LKB1, which can be helpful for better understanding of this molecular and its significance in cancers.

  2. Regulation of the Target of Rapamycin and Other Phosphatidylinositol 3-Kinase-Related Kinases by Membrane Targeting

    Directory of Open Access Journals (Sweden)

    Maristella De Cicco

    2015-09-01

    Full Text Available Phosphatidylinositol 3-kinase-related kinases (PIKKs play vital roles in the regulation of cell growth, proliferation, survival, and consequently metabolism, as well as in the cellular response to stresses such as ionizing radiation or redox changes. In humans six family members are known to date, namely mammalian/mechanistic target of rapamycin (mTOR, ataxia-telangiectasia mutated (ATM, ataxia- and Rad3-related (ATR, DNA-dependent protein kinase catalytic subunit (DNA-PKcs, suppressor of morphogenesis in genitalia-1 (SMG-1, and transformation/transcription domain-associated protein (TRRAP. All fulfill rather diverse functions and most of them have been detected in different cellular compartments including various cellular membranes. It has been suggested that the regulation of the localization of signaling proteins allows for generating a locally specific output. Moreover, spatial partitioning is expected to improve the reliability of biochemical signaling. Since these assumptions may also be true for the regulation of PIKK function, the current knowledge about the regulation of the localization of PIKKs at different cellular (membrane compartments by a network of interactions is reviewed. Membrane targeting can involve direct lipid-/membrane interactions as well as interactions with membrane-anchored regulatory proteins, such as, for example, small GTPases, or a combination of both.

  3. Checkpoint Kinases Regulate a Global Network of Transcription Factors in Response to DNA Damage

    Directory of Open Access Journals (Sweden)

    Eric J. Jaehnig

    2013-07-01

    Full Text Available DNA damage activates checkpoint kinases that induce several downstream events, including widespread changes in transcription. However, the specific connections between the checkpoint kinases and downstream transcription factors (TFs are not well understood. Here, we integrate kinase mutant expression profiles, transcriptional regulatory interactions, and phosphoproteomics to map kinases and downstream TFs to transcriptional regulatory networks. Specifically, we investigate the role of the Saccharomyces cerevisiae checkpoint kinases (Mec1, Tel1, Chk1, Rad53, and Dun1 in the transcriptional response to DNA damage caused by methyl methanesulfonate. The result is a global kinase-TF regulatory network in which Mec1 and Tel1 signal through Rad53 to synergistically regulate the expression of more than 600 genes. This network involves at least nine TFs, many of which have Rad53-dependent phosphorylation sites, as regulators of checkpoint-kinase-dependent genes. We also identify a major DNA damage-induced transcriptional network that regulates stress response genes independently of the checkpoint kinases.

  4. 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 ...

  5. Salt-inducible kinase 1 regulates E-cadherin expression and intercellular junction stability

    OpenAIRE

    Eneling, Kristina; Brion, Laura; Pinto, Vanda; Pinho, Maria J.; Sznajder, Jacob I.; Mochizuki , Naoki; Emoto, Kazuo; Soares-da-Silva, Patricio; Bertorello, Alejandro M.

    2012-01-01

    The protein kinase liver kinase B1 (LKB1) regulates cell polarity and intercellular junction stability. Also, LKB1 controls the activity of salt-inducible kinase 1 (SIK1). The role and relevance of SIK1 and its downstream effectors in linking the LKB1 signals within these processes are partially understood. We hypothesize that SIK1 may link LKB1 signals to the maintenance of epithelial junction stability by regulating E-cadherin expression. Results from our studies using a mouse lung alveolar...

  6. The Rho kinases I and II regulate different aspects of myosin II activity

    DEFF Research Database (Denmark)

    Yoneda, Atsuko; Multhaupt, Hinke A B; Couchman, John R

    2005-01-01

    The homologous mammalian rho kinases (ROCK I and II) are assumed to be functionally redundant, based largely on kinase construct overexpression. As downstream effectors of Rho GTPases, their major substrates are myosin light chain and myosin phosphatase. Both kinases are implicated in microfilament...... persistent ROCK II and guanine triphosphate-bound RhoA. In contrast, the microfilament cytoskeleton was enhanced by ROCK II down-regulation. Phagocytic uptake of fibronectin-coated beads was strongly down-regulated in ROCK II-depleted cells but not those lacking ROCK I. These effects originated in part from...

  7. 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.

  8. Ste20-related proline/alanine-rich kinase: A novel regulator of intestinal inflammation

    Institute of Scientific and Technical Information of China (English)

    Yutao Yan; Didier Merlin

    2008-01-01

    Recently, inflammatory bowel disease (IBD) has been the subject of considerable research, with increasing attention being paid to the loss of intestinal epithelial cell barrier function as a mechanism of pathogenesis. Ste20-related proline/alanine-rich kinase (SPAK) is involved in regulating barrier function. SPAK is known to interact with inflammation-related kinases (such as p38, JNK, NKCC1, PKCθ, WNK and MLCK), and with transcription factor AP-1, resulting in diverse biological phenomena, including cell differentiation, cell transformation and proliferation, cytoskeleton rearrangement, and regulation of chloride transport. This review examines the involvement of Ste20-like kinases and downstream mitogen-activated protein kinases (MAPKs) pathways in the pathogenesis and control of intestinal inflammation. The primary focus will be on the molecular features of intestinal inflammation, with an emphasis on the interaction between SPAK and other molecules, and the effect of these interactions on homeostatic maintenance, cell volume regulation and increased cell permeability in intestinal inflammation.

  9. Identification of novel pheromone-response regulators through systematic overexpression of 120 protein kinases in yeast.

    Science.gov (United States)

    Burchett, S A; Scott, A; Errede, B; Dohlman, H G

    2001-07-13

    Protein kinases are well known to transmit and regulate signaling pathways. To identify additional regulators of the pheromone signaling apparatus in yeast, we evaluated an array of 120 likely protein kinases encoded by the yeast genome. Each kinase was fused to glutathione S-transferase, overexpressed, and tested for changes in pheromone responsiveness in vivo. As expected, several known components of the pathway (YCK1, STE7, STE11, FUS3, and KSS1) impaired the growth arrest response. Seven other kinases also interfered with pheromone-induced growth arrest; in rank order they are as follows: YKL116c (renamed PRR1) = YDL214c (renamed PRR2) > YJL141c (YAK1, SRA1) > YNR047w = YCR091w (KIN82) = YIL095w (PRK1) > YCL024w (KCC4). Inhibition of pheromone signaling by PRR1, but not PRR2, required the glutathione S-transferase moiety. Both kinases inhibited gene transcription after stimulation with pheromone, a constitutively active kinase mutant STE11-4, or overexpression of the transcription factor STE12. Neither protein altered the ability of the mitogen-activated protein kinase (MAPK) Fus3 to feedback phosphorylate a known substrate, the MAPK kinase Ste7. These results reveal two new components of the pheromone-signaling cascade in yeast, each acting at a point downstream of the MAPK. PMID:11337509

  10. 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.

  11. Integrated stress response of vertebrates is regulated by four eIF2α kinases

    Science.gov (United States)

    Taniuchi, Shusuke; Miyake, Masato; Tsugawa, Kazue; Oyadomari, Miho; Oyadomari, Seiichi

    2016-01-01

    The integrated stress response (ISR) is a cytoprotective pathway initiated upon phosphorylation of the eukaryotic translation initiation factor 2 (eIF2α) residue designated serine-51, which is critical for translational control in response to various stress conditions. Four eIF2α kinases, namely heme-regulated inhibitor (HRI), protein kinase R (PKR), PKR-like endoplasmic reticulum kinase, (PERK) and general control non-depressible 2 (GCN2), have been identified thus far, and they are known to be activated by heme depletion, viral infection, endoplasmic reticulum stress, and amino acid starvation, respectively. Because eIF2α is phosphorylated under various stress conditions, the existence of an additional eIF2α kinase has been suggested. To validate the existence of the unidentified eIF2α kinase, we constructed an eIF2α kinase quadruple knockout cells (4KO cells) in which the four known eIF2α kinase genes were deleted using the CRISPR/Cas9-mediated genome editing. Phosphorylation of eIF2α was completely abolished in the 4KO cells by various stress stimulations. Our data suggests that the four known eIF2α kinases are sufficient for ISR and that there are no additional eIF2α kinases in vertebrates. PMID:27633668

  12. Integrated stress response of vertebrates is regulated by four eIF2α kinases.

    Science.gov (United States)

    Taniuchi, Shusuke; Miyake, Masato; Tsugawa, Kazue; Oyadomari, Miho; Oyadomari, Seiichi

    2016-01-01

    The integrated stress response (ISR) is a cytoprotective pathway initiated upon phosphorylation of the eukaryotic translation initiation factor 2 (eIF2α) residue designated serine-51, which is critical for translational control in response to various stress conditions. Four eIF2α kinases, namely heme-regulated inhibitor (HRI), protein kinase R (PKR), PKR-like endoplasmic reticulum kinase, (PERK) and general control non-depressible 2 (GCN2), have been identified thus far, and they are known to be activated by heme depletion, viral infection, endoplasmic reticulum stress, and amino acid starvation, respectively. Because eIF2α is phosphorylated under various stress conditions, the existence of an additional eIF2α kinase has been suggested. To validate the existence of the unidentified eIF2α kinase, we constructed an eIF2α kinase quadruple knockout cells (4KO cells) in which the four known eIF2α kinase genes were deleted using the CRISPR/Cas9-mediated genome editing. Phosphorylation of eIF2α was completely abolished in the 4KO cells by various stress stimulations. Our data suggests that the four known eIF2α kinases are sufficient for ISR and that there are no additional eIF2α kinases in vertebrates. PMID:27633668

  13. Tomato thymidine kinase is subject to inefficient TTP feedback regulation

    DEFF Research Database (Denmark)

    Larsen, Nicolai Balle; Munch-Petersen, Birgitte; Piskur, Jure

    2014-01-01

    A promising suicide gene therapy system to treat gliomas has been reported: the thymidine kinase 1 from tomato (toTK1) combined with the nucleoside analog pro-drug zidovudine (azidothymidine, AZT), which is known to penetrate the blood–brain barrier. Transduction with toTK1 has been found...

  14. 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

  15. Regulative Function of Telomerase and Extracelluar Regulated Protein Kinases to Leukemic Cell Apoptosis

    Institute of Scientific and Technical Information of China (English)

    李登举; 张瑶珍; 曹文静; 孙岚; 徐慧珍; 路武

    2002-01-01

    Summary: In order to investigate the regulative function of telomerase and phosphorylated (acti-vated) extracelluar regulated protein kinase (ERK) i and 2 in the leukemic cell lines HL-60 andK562 proliferation inhibition and apoptosis, three chemotherapeutic drugs Harringtonine (HRT),Vincristine(VCR)and Etoposide(Vp16)were selected as inducers. The proliferation inhibition ratewas detected by MTT method, the cell cycle and cell apoptosis was analyzed by flow cytometryand the telomerase activity was detected by the telomeric repeat amplification protocol (TRAP)assay and bioluminescence analysis method. The phosphorylated ERK1/2 protein expression wasdetected by western blot method. The results showed that HRT, VCR and Vp16 could inhibit cellproliferation, induce apoptosis, inhibit telomerase activity and down-regulate the protein expres-sion of phosphorylated ERK. It was suggested that ERK signal transduction pathway was involvedin the down-regulation of telomerase activity and the onset of apoptosis in the leukemic cells treat-ed by HRT, VCR and Vp16.

  16. Proteomic and functional genomic landscape of receptor tyrosine kinase and ras to extracellular signal-regulated kinase signaling.

    Science.gov (United States)

    Friedman, Adam A; Tucker, George; Singh, Rohit; Yan, Dong; Vinayagam, Arunachalam; Hu, Yanhui; Binari, Richard; Hong, Pengyu; Sun, Xiaoyun; Porto, Maura; Pacifico, Svetlana; Murali, Thilakam; Finley, Russell L; Asara, John M; Berger, Bonnie; Perrimon, Norbert

    2011-10-25

    Characterizing the extent and logic of signaling networks is essential to understanding specificity in such physiological and pathophysiological contexts as cell fate decisions and mechanisms of oncogenesis and resistance to chemotherapy. Cell-based RNA interference (RNAi) screens enable the inference of large numbers of genes that regulate signaling pathways, but these screens cannot provide network structure directly. We describe an integrated network around the canonical receptor tyrosine kinase (RTK)-Ras-extracellular signal-regulated kinase (ERK) signaling pathway, generated by combining parallel genome-wide RNAi screens with protein-protein interaction (PPI) mapping by tandem affinity purification-mass spectrometry. We found that only a small fraction of the total number of PPI or RNAi screen hits was isolated under all conditions tested and that most of these represented the known canonical pathway components, suggesting that much of the core canonical ERK pathway is known. Because most of the newly identified regulators are likely cell type- and RTK-specific, our analysis provides a resource for understanding how output through this clinically relevant pathway is regulated in different contexts. We report in vivo roles for several of the previously unknown regulators, including CG10289 and PpV, the Drosophila orthologs of two components of the serine/threonine-protein phosphatase 6 complex; the Drosophila ortholog of TepIV, a glycophosphatidylinositol-linked protein mutated in human cancers; CG6453, a noncatalytic subunit of glucosidase II; and Rtf1, a histone methyltransferase.

  17. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    Energy Technology Data Exchange (ETDEWEB)

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M.H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric (Van Andel); (Scripps); (NWU); (Purdue); (UCR); (Chinese Aca. Sci.); (NU Singapore)

    2014-10-02

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.

  18. 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...

  19. Arabidopsis map kinase 4 negatively regulates systemic acquired resistance

    DEFF Research Database (Denmark)

    Brodersen, P; Johansen, Bo; Petersen, M;

    2000-01-01

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

  20. Arabidopsis MAP kinase 4 negatively regulates systemic acquired resistance

    DEFF Research Database (Denmark)

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

    2000-01-01

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

  1. Regulation of Sphingolipid Biosynthesis by the Morphogenesis Checkpoint Kinase Swe1.

    Science.gov (United States)

    Chauhan, Neha; Han, Gongshe; Somashekarappa, Niranjanakumari; Gable, Kenneth; Dunn, Teresa; Kohlwein, Sepp D

    2016-01-29

    Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1, regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results, we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis, and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle. PMID:26634277

  2. Regulation of Sphingolipid Biosynthesis by the Morphogenesis Checkpoint Kinase Swe1*

    Science.gov (United States)

    Chauhan, Neha; Han, Gongshe; Somashekarappa, Niranjanakumari; Gable, Kenneth; Dunn, Teresa; Kohlwein, Sepp D.

    2016-01-01

    Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1, regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results, we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis, and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle. PMID:26634277

  3. Brf1 posttranscriptionally regulates pluripotency and differentiation responses downstream of Erk MAP kinase

    OpenAIRE

    Tan, Frederick E.; Elowitz, Michael B.

    2014-01-01

    AU-rich element mRNA-binding proteins (AUBPs) are key regulators of development, but how they are controlled and what functional roles they play depends on cellular context. Here, we show that Brf1 (zfp36l1), an AUBP from the Zfp36 protein family, operates downstream of FGF/Erk MAP kinase signaling to regulate pluripotency and cell fate decision making in mouse embryonic stem cells (mESCs). FGF/Erk MAP kinase signaling up-regulates Brf1, which disrupts the expression of core pluripotency-asso...

  4. Serum and glucocorticoid-regulated kinase 1 regulates neutrophil clearance during inflammation resolution.

    Science.gov (United States)

    Burgon, Joseph; Robertson, Anne L; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R; Walker, Paul; Hoggett, Emily E; Ward, Jonathan R; Farrow, Stuart N; Zuercher, William J; Jeffrey, Philip; Savage, Caroline O; Ingham, Philip W; Hurlstone, Adam F; Whyte, Moira K B; Renshaw, Stephen A

    2014-02-15

    The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid-regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease. PMID:24431232

  5. Serum and Glucocorticoid Regulated Kinase 1 (SGK1) Regulates Neutrophil Clearance During Inflammation Resolution

    Science.gov (United States)

    Burgon, Joseph; Robertson, Anne L.; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R.; Walker, Paul; Hoggett, Emily E.; Ward, Jonathan R.; Farrow, Stuart N.; Zuercher, William J.; Jeffrey, Philip; Savage, Caroline O.; Ingham, Philip W.; Hurlstone, Adam F.; Whyte, Moira K. B.; Renshaw, Stephen A.

    2013-01-01

    The inflammatory response is integral to maintaining health, by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralise invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein Serum and Glucocorticoid Regulated Kinase 1 (SGK1). We have characterised the expression patterns and regulation of SGK family members in human neutrophils, and shown that inhibition of SGK activity completely abrogates the anti-apoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signalling, and thus may prove a valuable therapeutic target for the treatment of inflammatory disease. PMID:24431232

  6. Serum and glucocorticoid-regulated kinase 1 regulates neutrophil clearance during inflammation resolution.

    Science.gov (United States)

    Burgon, Joseph; Robertson, Anne L; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R; Walker, Paul; Hoggett, Emily E; Ward, Jonathan R; Farrow, Stuart N; Zuercher, William J; Jeffrey, Philip; Savage, Caroline O; Ingham, Philip W; Hurlstone, Adam F; Whyte, Moira K B; Renshaw, Stephen A

    2014-02-15

    The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid-regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease.

  7. Role of Intrinsic and Extrinsic Factors in the Regulation of the Mitotic Checkpoint Kinase Bub1.

    Directory of Open Access Journals (Sweden)

    Claudia Breit

    Full Text Available The spindle assembly checkpoint (SAC monitors microtubule attachment to kinetochores to ensure accurate sister chromatid segregation during mitosis. The SAC members Bub1 and BubR1 are paralogs that underwent significant functional specializations during evolution. We report an in-depth characterization of the kinase domains of Bub1 and BubR1. BubR1 kinase domain binds nucleotides but is unable to deliver catalytic activity in vitro. Conversely, Bub1 is an active kinase regulated by intra-molecular phosphorylation at the P+1 loop. The crystal structure of the phosphorylated Bub1 kinase domain illustrates a hitherto unknown conformation of the P+1 loop docked into the active site of the Bub1 kinase. Both Bub1 and BubR1 bind Bub3 constitutively. A hydrodynamic characterization of Bub1:Bub3 and BubR1:Bub3 demonstrates both complexes to have 1:1 stoichiometry, with no additional oligomerization. Conversely, Bub1:Bub3 and BubR1:Bub3 combine to form a heterotetramer. Neither BubR1:Bub3 nor Knl1, the kinetochore receptor of Bub1:Bub3, modulate the kinase activity of Bub1 in vitro, suggesting autonomous regulation of the Bub1 kinase domain. We complement our study with an analysis of the Bub1 substrates. Our results contribute to the mechanistic characterization of a crucial cell cycle checkpoint.

  8. Protein kinase D2 induces invasion of pancreatic cancer cells by regulating matrix metalloproteinases

    OpenAIRE

    Wille, Christoph; Köhler, Conny; Armacki, Milena; Jamali, Arsia; Gössele, Ulrike; Pfizenmaier, Klaus; Seufferlein, Thomas; Eiseler, Tim

    2014-01-01

    Pancreatic cancer cell invasion, metastasis, and angiogenesis are major challenges for the development of novel therapeutic strategies. Protein kinase D (PKD) isoforms are involved in controlling tumor cell motility, angiogenesis, and metastasis. In particular PKD2 expression is up-regulated in pancreatic cancer, whereas PKD1 expression is lowered. We report that both kinases control pancreatic cancer cell invasive properties in an isoform-specific manner. PKD2 enhances invasion in three-dime...

  9. ZAP-70 kinase regulates HIV cell-to-cell spread and virological synapse formation

    OpenAIRE

    Sol-Foulon, Nathalie; Sourisseau, Marion; Porrot, Françoise; Thoulouze, Maria-Isabel; Trouillet, Céline; Nobile, Cinzia; Blanchet, Fabien; Di Bartolo, Vincenzo; Noraz, Nelly; Taylor, Naomi; Alcover, Andres; Hivroz, Claire; Schwartz, Olivier

    2007-01-01

    HIV efficiently spreads in lymphocytes, likely through virological synapses (VSs). These cell–cell junctions share some characteristics with immunological synapses, but cellular proteins required for their constitution remain poorly characterized. We have examined here the role of ZAP-70, a key kinase regulating T-cell activation and immunological synapse formation, in HIV replication. In lymphocytes deficient for ZAP-70, or expressing a kinase-dead mutant of the protein, HIV replication was ...

  10. 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...

  11. 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...

  12. Diacylglycerol Kinase-ζ Localization in Skeletal Muscle Is Regulated by Phosphorylation and Interaction with Syntrophins

    OpenAIRE

    Abramovici, Hanan; Hogan, Angela B.; Obagi, Christopher; Topham, Matthew K.; Gee, Stephen H.

    2003-01-01

    Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-ζ (DGK-ζ), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-ζ form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-ζ MARCKS domain. DGK-ζ mutants that do not ...

  13. PINCH proteins regulate cardiac contractility by modulating integrin-linked kinase-protein kinase B signaling.

    Science.gov (United States)

    Meder, Benjamin; Huttner, Inken G; Sedaghat-Hamedani, Farbod; Just, Steffen; Dahme, Tillman; Frese, Karen S; Vogel, Britta; Köhler, Doreen; Kloos, Wanda; Rudloff, Jessica; Marquart, Sabine; Katus, Hugo A; Rottbauer, Wolfgang

    2011-08-01

    Integrin-linked kinase (ILK) is an essential component of the cardiac mechanical stretch sensor and is bound in a protein complex with parvin and PINCH proteins, the so-called ILK-PINCH-parvin (IPP) complex. We have recently shown that inactivation of ILK or β-parvin activity leads to heart failure in zebrafish via reduced protein kinase B (PKB/Akt) activation. Here, we show that PINCH proteins localize at sarcomeric Z disks and costameres in the zebrafish heart and skeletal muscle. To investigate the in vivo role of PINCH proteins for IPP complex stability and PKB signaling within the vertebrate heart, we inactivated PINCH1 and PINCH2 in zebrafish. Inactivation of either PINCH isoform independently leads to instability of ILK, loss of stretch-responsive anf and vegf expression, and progressive heart failure. The predominant cause of heart failure in PINCH morphants seems to be loss of PKB activity, since PKB phosphorylation at serine 473 is significantly reduced in PINCH-deficient hearts and overexpression of constitutively active PKB reconstitutes cardiac function in PINCH morphants. These findings highlight the essential function of PINCH proteins in controlling cardiac contractility by granting IPP/PKB-mediated signaling.

  14. 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.

  15. Post-transcriptional regulation of the chicken thymidine kinase gene.

    Science.gov (United States)

    Groudine, M; Casimir, C

    1984-02-10

    In attempting to understand the molecular basis of the control of chicken thymidine kinase (cTK) gene expression, we have examined the steady state cTK RNA content, and the patterns of DNA methylation, chromatin structure and endogenous nuclear runoff transcription of this gene in dividing and non-dividing cells. Our results reveal that the steady state level of cTK poly A+ RNA is correlated with the divisional activity of normal avian cells and tissues. However, no differences in the pattern of Hpa II site methylation or chromatin structure are found among cells containing high or undetectable levels of steady state cTK RNA. In addition, no differences in cTK transcription as assayed by nuclear runoff experiments are detectable in isolated nuclei derived from dividing or non-dividing cells containing high or low levels of steady state cTK RNA. These results suggest that the principal control of chicken thymidine kinase gene expression is post-transcriptional in nature.

  16. Post-transcriptional regulation of the chicken thymidine kinase gene.

    Science.gov (United States)

    Groudine, M; Casimir, C

    1984-02-10

    In attempting to understand the molecular basis of the control of chicken thymidine kinase (cTK) gene expression, we have examined the steady state cTK RNA content, and the patterns of DNA methylation, chromatin structure and endogenous nuclear runoff transcription of this gene in dividing and non-dividing cells. Our results reveal that the steady state level of cTK poly A+ RNA is correlated with the divisional activity of normal avian cells and tissues. However, no differences in the pattern of Hpa II site methylation or chromatin structure are found among cells containing high or undetectable levels of steady state cTK RNA. In addition, no differences in cTK transcription as assayed by nuclear runoff experiments are detectable in isolated nuclei derived from dividing or non-dividing cells containing high or low levels of steady state cTK RNA. These results suggest that the principal control of chicken thymidine kinase gene expression is post-transcriptional in nature. PMID:6199739

  17. Corticosterone Regulates pERK1/2 Map Kinase in a Chronic Depression Model

    OpenAIRE

    Gourley, Shannon L.; Wu, Florence J.; Taylor, Jane R.

    2008-01-01

    Neurotransmitter- or neurotrophin-regulated intracellular signaling in the hippocampus is hypothesized to contribute to depression and antidepressant (ADT) efficacy. Extracellular signal-regulated kinase 1/2 (ERK1/2) is downstream of several receptor types and regulates transcriptional activity of many targets; ERK1/2 may thereby influence mood and affect. Using a novel, ADT-sensitive depression model in mice, we show that prior corticosterone exposure decreases motivated behavior, sucrose co...

  18. Regulation of MAP kinase signaling cascade by microRNAs in Oryza sativa

    OpenAIRE

    Raghuram, Badmi; Sheikh, Arsheed Hussain; Sinha, Alok Krishna

    2014-01-01

    Mitogen activated protein kinase (MAPK) pathway is one of the most conserved signaling cascade in plants regulating a plethora of cellular processes including normal growth and development, abiotic and biotic stress responses. The perception of external cues triggers the phosphorylation of three tier MAPKKK-MAPKK-MAPK cascade which finally modifies a downstream substrate thereby regulating the cellular processes. Whereas, the transcription regulation by MAPKs, mediated through their substrate...

  19. An Integrative Approach for the Large-scale Identification of Human Genome Kinases Regulating Cancer Metastasis

    Science.gov (United States)

    Zhang, Hanshuo; Wu, Pu-Yen; Ma, Ming; Ye, Yanzheng; Hao, Yang; Yang, Junyu; Yin, Shenyi; Sun, Changhong; Phan, John H.; Wang, May D.; Xi, Jianzhong Jeff

    2016-01-01

    Kinases regulate the majority of biological processes and become one of important groups of drug targets. To identify more kinases being potential for cancer therapy, we developed an integrative approach for the large-scale screen of functional genes capable of regulating the main traits of cancer metastasis, including cell migration as well as invasion. We first employed self-assembled cell microarray (SAMcell) to screen functional genes that regulate cancer cell migration using a siRNA library targeting 710 human genome kinase genes. We identified 81 genes capable of significantly regulating cancer cell migration. Following with invasion assays and bio-informatics analysis, we discovered that 16 genes with differentially expression in cancer samples can regulate both cell migration and invasion, among which 10 genes have been well known to play critical roles in the cancer development. The remaining 6 genes were experimentally validated to have the capacities of regulating the metastasis-related traits, including cell proliferation, apoptosis and anoikis activities besides cell motility. Together, these findings provide a new insight into the therapeutic use of human kinases. PMID:23751374

  20. mTOR independent regulation of macroautophagy by Leucine Rich Repeat Kinase 2 via Beclin-1

    Science.gov (United States)

    Manzoni, Claudia; Mamais, Adamantios; Roosen, Dorien A.; Dihanich, Sybille; Soutar, Marc P. M.; Plun-Favreau, Helene; Bandopadhyay, Rina; Hardy, John; Tooze, Sharon A.; Cookson, Mark R.; Lewis, Patrick A.

    2016-01-01

    Leucine rich repeat kinase 2 is a complex enzyme with both kinase and GTPase activities, closely linked to the pathogenesis of several human disorders including Parkinson’s disease, Crohn’s disease, leprosy and cancer. LRRK2 has been implicated in numerous cellular processes; however its physiological function remains unclear. Recent reports suggest that LRRK2 can act to regulate the cellular catabolic process of macroautophagy, although the precise mechanism whereby this occurs has not been identified. To investigate the signalling events through which LRRK2 acts to influence macroautophagy, the mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) and Beclin-1/phosphatidylinositol 3-kinase (PI3K) pathways were evaluated in astrocytic cell models in the presence and absence of LRRK2 kinase inhibitors. Chemical inhibition of LRRK2 kinase activity resulted in the stimulation of macroautophagy in a non-canonical fashion, independent of mTOR and ULK1, but dependent upon the activation of Beclin 1-containing class III PI3-kinase. PMID:27731364

  1. Cell cycle-dependent regulation of Aurora kinase B mRNA by the Microprocessor complex.

    Science.gov (United States)

    Jung, Eunsun; Seong, Youngmo; Seo, Jae Hong; Kwon, Young-Soo; Song, Hoseok

    2014-03-28

    Aurora kinase B regulates the segregation of chromosomes and the spindle checkpoint during mitosis. In this study, we showed that the Microprocessor complex, which is responsible for the processing of the primary transcripts during the generation of microRNAs, destabilizes the mRNA of Aurora kinase B in human cells. The Microprocessor-mediated cleavage kept Aurora kinase B at a low level and prevented premature entrance into mitosis. The cleavage was reduced during mitosis leading to the accumulation of Aurora kinase B mRNA and protein. In addition to Aurora kinase B mRNA, the processing of other primary transcripts of miRNAs were also decreased during mitosis. We found that the cleavage was dependent on an RNA helicase, DDX5, and the association of DDX5 and DDX17 with the Microprocessor was reduced during mitosis. Thus, we propose a novel mechanism by which the Microprocessor complex regulates stability of Aurora kinase B mRNA and cell cycle progression.

  2. 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

  3. Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

    Science.gov (United States)

    Boo, Yong Chool; Jo, Hanjoong

    2003-01-01

    Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca2+/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases-protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases-are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation.

  4. Function and regulation of Aurora/Ipl1p kinase family in cell division

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    During mitosis, the parent cell distributes its genetic materials equally into two daughter cells through chromosome segregation, a complex movements orchestrated by mitotic kinases and its effector proteins.Faithful chromosome segregation and cytokinesis ensure that each daughter cell receives a full copy of genetic materials of parent cell. Defects in these processes can lead to aneuploidy or polyploidy. Aurora/Ipl1p fanily,a class of conserved serine/threonine kinases, plays key roles in chromosome segregation and cytokinesis.This article highlights the function and regulation of Aurora/Ipl1p family in mitosis and provides potential links between aberrant regulation of Aurora/Ipl1p kinases and pathogenesis of human cancer.

  5. 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...

  6. SOCS proteins in regulation of receptor tyrosine kinase signaling

    DEFF Research Database (Denmark)

    Kazi, Julhash U.; Kabir, Nuzhat N.; Flores Morales, Amilcar;

    2014-01-01

    signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar...... proteins, SOCS1-7, and cytokine-inducible SH2-containing protein (CIS). A key feature of this family of proteins is the presence of an SH2 domain and a SOCS box. Recent studies suggest that SOCS proteins also play a role in RTK signaling. Activation of RTK results in transcriptional activation of SOCS......-encoding genes. These proteins associate with RTKs through their SH2 domains and subsequently recruit the E3 ubiquitin machinery through the SOCS box, and thereby limit receptor stability by inducing ubiquitination. In a similar fashion, SOCS proteins negatively regulate mitogenic signaling by RTKs. It is also...

  7. 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.

  8. AGC kinases regulate phosphorylation and activation of eukaryotic translation initiation factor 4B

    NARCIS (Netherlands)

    van Gorp, A. G. M.; van der Vos, K. E.; Brenkman, A. B.; Bremer, A.; van den Broek, N.; Zwartkruis, F.; Hershey, J. W.; Burgering, B. M. T.; Calkhoven, C. F.; Coffer, P. J.

    2009-01-01

    Eukaryotic translation initiation factor 4B (eIF4B) plays a critical role during the initiation of protein synthesis and its activity can be regulated by multiple phosphorylation events. In a search for novel protein kinase B (PKB/c-akt) substrates, we identified eIF4B as a potential target. Using a

  9. An adenylate kinase is involved in KATP channel regulation of mouse pancreatic beta cells.

    NARCIS (Netherlands)

    Schulze, D.U.; Dufer, M.; Wieringa, B.; Krippeit-Drews, P.; Drews, G.

    2007-01-01

    AIMS/HYPOTHESIS: In a previous study, we demonstrated that a creatine kinase (CK) modulates K(ATP) channel activity in pancreatic beta cells. To explore phosphotransfer signalling pathways in more detail, we examined whether K(ATP) channel regulation in beta cells is determined by a metabolic intera

  10. Regulation of Polyphosphate Kinase Production by Antisense RNA in Pseudomonas fluorescens Pf0-1

    OpenAIRE

    Silby, Mark W.; Julie S Nicoll; Levy, Stuart B.

    2012-01-01

    Pseudomonas spp. adapt rapidly to environmental fluctuations. Loss or overproduction of polyphosphate reduces the fitness of Pseudomonas fluorescens Pf0-1, indicating the importance of the fine-tuning of polyphosphate production. An antisense RNA was investigated and shown to regulate the polyphosphate kinase gene (ppk) by a posttranscriptional mechanism reducing ppk transcript abundance.

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    Arabidopsis MAP kinase 4 (MPK4) functions as a regulator of pathogen defense responses, because it is required for both repression of salicylic acid (SA)-dependent resistance and for activation of jasmonate (JA)-dependent defense gene expression. To understand MPK4 signaling mechanisms, we used...

  12. Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein Synthesis

    Directory of Open Access Journals (Sweden)

    Kimon C. Kanelakis

    2009-01-01

    Full Text Available The heme-regulated inhibitor (HRI negatively regulates protein synthesis by phosphorylating eukaryotic initiation factor-2α (eIF2α thereby inhibiting protein translation. The importance of HRI in regulating hemoglobin synthesis in erythroid cells makes it an attractive molecular target in need of further characterization. In this work, we have cloned and expressed the canine form of the HRI kinase. The canine nucleotide sequence has 86%, 82%, and 81% identity to the human, mouse, and rat HRI, respectively. It was noted that an isoleucine residue in the ATP binding site of human, rat, and mouse HRI is replaced by a valine in the canine kinase. The expression of canine HRI protein by in vitro translation using wheat germ lysate or in Sf9 cells using a baculovirus expression system was increased by the addition of hemin. Following purification, the canine protein was found to be 72 kD and showed kinase activity determined by its ability to phosphorylate a synthetic peptide substrate. Quercetin, a kinase inhibitor known to inhibit mouse and human HRI, inhibits canine HRI in a concentration-dependent manner. Additionally, quercetin is able to increase de novo protein synthesis in canine reticulocytes. We conclude that the canine is a suitable model species for studying the role of HRI in erythropoiesis.

  13. Proteins regulating cyclin dependent kinases Cdk4 and Cdk5

    NARCIS (Netherlands)

    Moorthamer, M.J.M.W.

    1999-01-01

    The exact passage through the eukaryotic cell cycle is regulated by the progressive activation and inactivation of a family Cdk-s. Cancer cells evolve from normal cells when some essential processes in a dividing cell malfunction. This causes inappropriate replication, segregation and repair of the

  14. The lipid kinase phosphatidylinositol-4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A.

    Directory of Open Access Journals (Sweden)

    Simon Reiss

    2013-05-01

    Full Text Available The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα is an essential host factor of hepatitis C virus (HCV replication. PI4KIIIα catalyzes the synthesis of phosphatidylinositol 4-phosphate (PI4P accumulating in HCV replicating cells due to enzyme activation resulting from its interaction with nonstructural protein 5A (NS5A. This study describes the interaction between PI4KIIIα and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in PI4KIIIα interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIIIα functional interaction site (PFIS encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIIIα binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIIIα. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58, indicating that PI4KIIIα affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIIIα or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIIIα increased relative abundance of basally phosphorylated NS5A (p56. PI4KIIIα therefore regulates the phosphorylation status of NS5A and viral RNA replication by favoring p56 or repressing p58 synthesis. Replication deficiencies of PFIS mutants in NS5A could not be rescued by increasing PI4P levels, but by supplying functional NS5A, supporting an essential role of PI4KIIIα in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIIIα activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIIIα in the morphogenesis

  15. Receptor tyrosine kinase signaling regulates replication of the peste des petits ruminants virus.

    Science.gov (United States)

    Chaudhary, K; Chaubey, K K; Singh, S V; Kumar, N

    2015-03-01

    In this study, we found out that blocking the receptor tyrosine kinase (RTK) signaling in Vero cells by tryphostin AG879 impairs the in vitro replication of the peste des petits ruminants virus (PPRV). A reduced virus replication in Trk1-knockdown (siRNA) Vero cells confirmed the essential role of RTK in the virus replication, in particular a specific regulation of viral RNA synthesis. These data represent the first evidence that the RTK signaling regulates replication of a morbillivirus. PMID:25790054

  16. Diacylglycerol kinase theta and zeta isoforms: regulation of activity, protein binding partners and physiological functions

    OpenAIRE

    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 Retinoblastoma tumour suppressor protein (pRB) and the pRB-related proteins p107 and p130, key regulators of the cell-cycle, differentiation and apoptosis. The interaction between pRB and DGKzeta is regulated ...

  17. 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.

  18. 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.

  19. c-Src regulates cell cycle proteins expression through protein kinase B/glycogen synthase kinase 3 beta and extracellular signal-regulated kinases 1/2 pathways in MCF-7 cells.

    Science.gov (United States)

    Liu, Xiang; Du, Liying; Feng, Renqing

    2013-07-01

    We have demonstrated that c-Src suppression inhibited the epithelial to mesenchymal transition in human breast cancer cells. Here, we investigated the role of c-Src on the cell cycle progression using siRNAs and small molecule inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Western blot analysis demonstrated the down-regulation of cyclin D1 and cyclin E and up-regulation of p27 Kip1 after c-Src suppression by PP2. Incubation of cells in the presence of PP2 significantly blocked the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), protein kinase B (AKT), and glycogen synthase kinase 3 beta (GSK3β). Specific pharmacological inhibitors of MEK1/2/ERK1/2 and phosphatidylinositide 3-kinase/AKT pathways were used to demonstrate the relationship between the signal cascade and cell cycle proteins expression. The expression of cyclin D1 and cyclin E were decreased after inhibition of ERK1/2 or AKT activity, whereas the p27 Kip1 expression was increased. In addition, knockdown of c-Src by siRNAs reduced cell proliferation and phosphorylation of ERK1/2, AKT, and GSK3β. After c-Src depletion by siRNAs, we observed significant down-regulation of cyclin D1 and cyclin E, and up-regulation of p27 Kip1. These results suggest that c-Src suppression by PP2 or siRNAs may regulate the progression of cell cycle through AKT/GSK3β and ERK1/2 pathways.

  20. Association of protein kinase FA/GSK-3alpha (a proline-directed kinase and a regulator of protooncogenes) with human cervical carcinoma dedifferentiation/progression.

    Science.gov (United States)

    Yang, S D; Yu, J S; Lee, T T; Ni, M H; Yang, C C; Ho, Y S; Tsen, T Z

    1995-10-01

    Computer analysis of protein phosphorylation-sites sequence revealed that most transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3alpha (kinase FA/GSK-3alpha) (a particular member of PDPK family) has been optimized for human cervical tissue and used to demonstrate for the first time significantly increased (P < 0.001) activity in poorly differentiated cervical carcinoma (82.8 +/- 6.6 U/mg of protein), moderately differentiated carcinoma (36.2 +/- 3.4 U/mg of protein), and well-differentiated carcinoma (18.3 +/- 2.4 U/mg of protein) from 36 human cervical carcinoma samples when compared to 12 normal controls (4.9 +/- 0.6 U/mg of protein). Immunoblotting analysis further revealed that increased activity of kinase FA/GSK-3alpha in cervical carcinoma is due to overexpression of protein synthesis of the kinase. Taken together, the results provide initial evidence that overexpression of protein synthesis and cellular activity of kinase FA/GSK-3alpha may be involved in human cervical carcinoma dedifferentiation/progression, supporting an association of proline-directed protein kinase with neoplastic transformation and tumorigenesis. Since protein kinase FA/GSK-3alpha may function as a possible regulator of transcription factors/proto-oncogenes, the results further suggest that kinase FA/GSK-3alpha may play a potential role in human cervical carcinogenesis, especially in its dedifferentiation and progression.

  1. 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

  2. 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...

  3. c-Src regulates cell cycle proteins expression through protein kinase B/glycogen synthase kinase 3 beta and extracellular signal-regulated kinases 1/2 pathways in MCF-7 cells

    Institute of Scientific and Technical Information of China (English)

    Xiang Liu; Liying Du; Renqing Feng

    2013-01-01

    We have demonstrated that c-Src suppression inhibited the epithelial to mesenchymal transition in human breast cancer cells.Here,we investigated the role of c-Src on the cell cycle progression using siRNAs and small molecule inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine (PP2).Western blot analysis demonstrated the downregulation of cyclin D1 and cyclin E and up-regulation of p27 Kip1 after c-Src suppression by PP2.Incubation of cells in the presence of PP2 significantly blocked the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2),protein kinase B (AKT),and glycogen synthase kinase 3 beta (GSK3β).Specific pharmacological inhibitors of MEK1/2/ERK1/2 and phosphatidylinositide 3-kinase/AKTpathways were used to demonstrate the relationship between the signal cascade and cell cycle proteins expression.The expression of cyclin D1 and cyclin E were decreased after inhibition of ERK1/2 or AKT activity,whereas the p27 Kip1 expression was increased.In addition,knockdown of c-Src by siRNAs reduced cell proliferation and phosphorylation of ERK1/2,AKT,and GSK3β.After c-Src depletion by siRNAs,we observed significant down-regulation of cyclin D1 and cyclin E,and up-regulation of p27 Kip1.These results suggest that c-Src suppression by PP2 or siRNAs may regulate the progression of cell cycle through AKT/GSK3β and ERK1/2 pathways.

  4. 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.

  5. Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

    Science.gov (United States)

    Liu, Z.; Xia, M.; Poovaiah, B. W.

    1998-01-01

    cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

  6. Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Vanesa Olivares-Illana

    2008-06-01

    Full Text Available Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.

  7. Casein Kinase 2 Regulates the mRNA-destabilizing Activity of Tristetraprolin*

    OpenAIRE

    Lee, Won Hyeok; Lee, Hyun Hee; Vo, Mai-Tram; Kim, Hyo Jeong; Ko, Myoung Seok; Im, Yeong-Cheol; Min, Young Joo; Lee, Byung Ju; Cho, Wha Ja; Park, Jeong Woo

    2011-01-01

    Tristetraprolin (TTP) is an AU-rich element-binding protein that regulates mRNA stability. We previously showed that TTP acts as a negative regulator of VEGF gene expression in colon cancer cells. The p38 MAPK pathway is known to suppress the TTP activity. However, until now the signaling pathway to enhance TTP function is not well known. Here, we show that casein kinase 2 (CK2) enhances the TTP function in the regulation of the VEGF expression in colon cancer cells. CK2 increased TTP protein...

  8. Transcriptome analysis of the Lactococcus lactis ArgR and AhrC regulons

    DEFF Research Database (Denmark)

    Larsen, Rasmus; van Hijum, Sacha A. F. T.; Martinussen, Jan;

    2008-01-01

    In previous studies, we have shown that direct protein-protein. interaction between the two regulators ArgR and AhrC in Lactococcus lactis is required for arginine-dependent repression of the biosynthetic argC promoter and the activation of the catabolic arcA promoter. Here, we establish the global...... ArgR and AhrC regulons by transcriptome analyses and show that both regulators are dedicated to the control of arginine metabolism in L. lactis....

  9. 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.

  10. Cancer metabolism meets systems biology: Pyruvate kinase isoform PKM2 is a metabolic master regulator

    Directory of Open Access Journals (Sweden)

    Fabian V Filipp

    2013-01-01

    Full Text Available Pyruvate kinase activity is controlled by a tightly woven regulatory network. The oncofetal isoform of pyruvate kinase (PKM2 is a master regulator of cancer metabolism. PKM2 engages in parallel, feed-forward, positive and negative feedback control contributing to cancer progression. Besides its metabolic role, non-metabolic functions of PKM2 as protein kinase and transcriptional coactivator for c-MYC and hypoxia-inducible factor 1-alpha are essential for epidermal growth factor receptor activation-induced tumorigenesis. These biochemical activities are controlled by a shift in the oligomeric state of PKM2 that includes acetylation, oxidation, phosphorylation, prolyl hydroxylation and sumoylation. Metabolically active PKM2 tetramer is allosterically regulated and responds to nutritional and stress signals. Metabolically inactive PKM2 dimer is imported into the nucleus and can function as protein kinase stimulating transcription. A systems biology approach to PKM2 at the genome, transcriptome, proteome, metabolome and fluxome level reveals how differences in biomolecular structure translate into a global rewiring of cancer metabolism. Cancer systems biology takes us beyond the Warburg effect, opening unprecedented therapeutic opportunities.

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

    Science.gov (United States)

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

    2014-10-27

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

  12. Negative Regulation of Receptor Tyrosine Kinase (RTK Signaling: A Developing Field

    Directory of Open Access Journals (Sweden)

    Fernanda Ledda

    2007-01-01

    Full Text Available ophic factors control cellular physiology by activating specific receptor tyrosine kinases (RTKs. While the over activation of RTK signaling pathways is associated with cell growth and cancer, recent findings support the concept that impaired down-regulation or deactivation of RTKs may also be a mechanism involved in tumor formation. Under this perspective, the molecular determinants of RTK signaling inhibition may act as tumor-suppressor genes and have a potential role as tumor markers to monitor and predict disease progression. Here, we review the current understanding of the physiological mechanisms that attenuate RTK signaling and discuss evidence that implicates deregulation of these events in cancer.Abbreviations: BDP1: Brain-derived phosphatase 1; Cbl: Casitas B-lineage lymphoma; CIN-85: Cbl-interacting protein of 85 kDa; DER: Drosophila EGFR; EGFR: Epidermal growth factor receptor; ERK 1/2: Extracellular signal-regulated kinase 1/2; Grb2: Growth factor receptor-bound protein 2; HER2: Human epidermal growth factor receptor 2; LRIG: Leucine-rich repeats and immunoglobulin-like domain 1; MAPK: Mitogen-activated protein kinase; Mig 6: Mitogen-inducible gene 6; PTEN: Phosphatase and tensin homologue; RET: Rearranged in transformation; RTK: Receptor tyrosine kinase. SH2 domain: Src-homology 2 domain; SH3 domain: Src-homology 3 domain; Spry: Sprouty.

  13. Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus.

    Science.gov (United States)

    Qiu, Jin-Long; Fiil, Berthe Katrine; Petersen, Klaus; Nielsen, Henrik Bjørn; Botanga, Christopher J; Thorgrimsen, Stephan; Palma, Kristoffer; Suarez-Rodriguez, Maria Cristina; Sandbech-Clausen, Signe; Lichota, Jacek; Brodersen, Peter; Grasser, Klaus D; Mattsson, Ole; Glazebrook, Jane; Mundy, John; Petersen, Morten

    2008-08-20

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

  14. Extracellular-regulated kinase 2 is activated by the enhancement of hinge flexibility.

    Science.gov (United States)

    Sours, Kevin M; Xiao, Yao; Ahn, Natalie G

    2014-05-01

    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 flexibility within the hinge region facilitates kinase activation. We show that hinge mutations enhancing flexibility promote changes in the nucleotide binding mode consistent with domain movement, without requiring phosphorylation. They also lead to the activation of monophosphorylated ERK2, a form that is normally inactive. The hinge mutations bypass the need for pTyr but not pThr, suggesting that Tyr phosphorylation controls hinge motions. In agreement, monophosphorylation of pTyr enhances both hinge flexibility and nucleotide binding mode, measured by hydrogen-exchange mass spectrometry. Our findings demonstrate that regulated protein motions underlie kinase activation. Our working model is that constraints to domain movement in ERK2 are overcome by phosphorylation at pTyr, which increases hinge dynamics to promote the active conformation of the catalytic site.

  15. 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...

  16. Cbl participates in shikonin-induced apoptosis by negatively regulating phosphoinositide 3-kinase/protein kinase B signaling.

    Science.gov (United States)

    Qu, Dan; Xu, Xiao-Man; Zhang, Meng; Jiang, Ting-Shu; Zhang, Yi; Li, Sheng-Qi

    2015-07-01

    Shikonin, a naturally occurring naphthoquinone, exhibits anti-tumorigenic activity. However, its precise mechanisms of action have remained elusive. In the present study, the involvement in the action of shikonin of the ubiquitin ligases Cbl-b and c-Cbl, which are negative regulators of phosphoinositide 3-kinase (PI3K) activation, was investigated. Shikonin was observed to reduce cell viability and induce apoptosis and G2/M phase arrest in lung cancer cells. In addition, shikonin increased the protein levels of B-cell lymphoma 2 (Bcl-2)-associated X and p53 and reduced those of Bcl-2. Additionally, shikonin inhibited PI3k/Akt activity and upregulated Cbl protein expression. In addition, a specific inhibitor of PI3K, LY294002, was observed to have a synergistic effect on the proliferation inhibition and apoptotic induction of A549 cells with shikonin. In conclusion, the results of the present study suggested that Cbl proteins promote shikonin-induced apoptosis by negatively regulating PI3K/Akt signaling in lung cancer cells.

  17. Protein Kinase D Enzymes as Regulators of EMT and Cancer Cell Invasion.

    Science.gov (United States)

    Durand, Nisha; Borges, Sahra; Storz, Peter

    2016-01-01

    The Protein Kinase D (PKD) isoforms PKD1, PKD2, and PKD3 are effectors of the novel Protein Kinase Cs (nPKCs) and diacylglycerol (DAG). PKDs impact diverse biological processes like protein transport, cell migration, proliferation, epithelial to mesenchymal transition (EMT) and apoptosis. PKDs however, have distinct effects on these functions. While PKD1 blocks EMT and cell migration, PKD2 and PKD3 tend to drive both processes. Given the importance of EMT and cell migration to the initiation and progression of various malignancies, abnormal expression of PKDs has been reported in multiple types of cancers, including breast, pancreatic and prostate cancer. In this review, we discuss how EMT and cell migration are regulated by PKD isoforms and the significance of this regulation in the context of cancer development. PMID:26848698

  18. Protein Kinase D Enzymes as Regulators of EMT and Cancer Cell Invasion

    Directory of Open Access Journals (Sweden)

    Nisha Durand

    2016-02-01

    Full Text Available The Protein Kinase D (PKD isoforms PKD1, PKD2, and PKD3 are effectors of the novel Protein Kinase Cs (nPKCs and diacylglycerol (DAG. PKDs impact diverse biological processes like protein transport, cell migration, proliferation, epithelial to mesenchymal transition (EMT and apoptosis. PKDs however, have distinct effects on these functions. While PKD1 blocks EMT and cell migration, PKD2 and PKD3 tend to drive both processes. Given the importance of EMT and cell migration to the initiation and progression of various malignancies, abnormal expression of PKDs has been reported in multiple types of cancers, including breast, pancreatic and prostate cancer. In this review, we discuss how EMT and cell migration are regulated by PKD isoforms and the significance of this regulation in the context of cancer development.

  19. Anaplastic Lymphoma Kinase Acts in the Drosophila Mushroom Body to Negatively Regulate Sleep.

    Directory of Open Access Journals (Sweden)

    Lei Bai

    2015-11-01

    Full Text Available Though evidence is mounting that a major function of sleep is to maintain brain plasticity and consolidate memory, little is known about the molecular pathways by which learning and sleep processes intercept. Anaplastic lymphoma kinase (Alk, the gene encoding a tyrosine receptor kinase whose inadvertent activation is the cause of many cancers, is implicated in synapse formation and cognitive functions. In particular, Alk genetically interacts with Neurofibromatosis 1 (Nf1 to regulate growth and associative learning in flies. We show that Alk mutants have increased sleep. Using a targeted RNAi screen we localized the negative effects of Alk on sleep to the mushroom body, a structure important for both sleep and memory. We also report that mutations in Nf1 produce a sexually dimorphic short sleep phenotype, and suppress the long sleep phenotype of Alk. Thus Alk and Nf1 interact in both learning and sleep regulation, highlighting a common pathway in these two processes.

  20. Amino acid microsequencing of internal tryptic peptides of heme-regulated eukaryotic initiation factor 2 alpha subunit kinase: homology to protein kinases.

    Science.gov (United States)

    Chen, J J; Pal, J K; Petryshyn, R; Kuo, I; Yang, J M; Throop, M S; Gehrke, L; London, I M

    1991-01-01

    We have purified the heme-regulated eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) kinase (HRI) from rabbit reticulocytes for amino acid microsequencing. This kinase is a single 92-kDa polypeptide and migrates in perfect alignment with 32P-labeled HRI on SDS/PAGE. Its functions of binding ATP and of autophosphorylation and eIF-2 alpha phosphorylation are inhibited by hemin. The amino acid sequences of three tryptic peptides of HRI have been obtained. A search of the data base of the National Biomedical Research Foundation reveals that these amino acid sequences are unique and that two of these three sequences show homology to protein kinases. HRI peptide P-52 contains Asp-Phe-Gly, which is the most highly conserved short stretch of amino acids in catalytic domain VII of protein kinases. HRI peptide P-74 contains the conserved amino acid residues Asp-(Met)-Tyr-Ser-(Val)-Gly-Val found in catalytic domain IX of protein kinases [Hanks, S. K., Quinn, A. M. & Hunter, T. (1988) Science 241, 42-52]. These findings are consistent with the autokinase and eIF-2 alpha kinase activities of HRI. Synthetic HRI peptide P-74 is a very potent inhibitor of eIF-2 alpha phosphorylation by HRI. Since little is known about the function of conserved domain IX, P-74 peptide may be useful in elucidating the role of this domain of protein kinases. Images PMID:1671169

  1. Novel Role and Regulation of the Interleukin-1 Receptor Associated Kinase (IRAK) Family Proteins

    Institute of Scientific and Technical Information of China (English)

    Yingsu Huang; Anna Misior; Liwu Li

    2005-01-01

    The interleukin-1 receptor associated kinases (IRAKs) sit at the bottle neck for the Toll-like-receptor (TLR) mediated signal transduction process controlling host innate immune response. However, the exact role and regulation of IRAKs are still in the early stage and not fully understood. This review intends to summarize the recent advancement in this important topic and points out areas that need further intensive investigation.

  2. Novel Role and Regulation of the Interleukin-1 Receptor Associated Kinase (IRAK) Family Proteins

    Institute of Scientific and Technical Information of China (English)

    YingsuHuang; AnnaMisior

    2005-01-01

    The interleukin-1 receptor associated kinases (IRAKs) sit at the bottle neck for the Toll-like-receptor (TLR) mediated signal transduction process controlling host innate immune response. However, the exact role and regulation of IRAKs are still in the early stage and not fully understood. This review intends to summarize the recent advancement in this important topic and points out areas that need further intensive investigation. Cellular & Molecular Immunology. 2005;2(1):36-39.

  3. Regulation of Androgen Receptor and Prostate Cancer Growth by Cyclin-dependent Kinase 5*

    OpenAIRE

    Hsu, Fu-Ning; Chen, Mei-Chih; Chiang, Ming-Ching; Lin, Eugene; Lee, Yueh-Tsung; Huang, Pao-Hsuan; Lee, Guan-Shun; Lin, Ho

    2011-01-01

    Prostate cancer is the most frequently diagnosed male malignancy. The normal prostate development and prostate cancer progression are mediated by androgen receptor (AR). Recently, the roles of cyclin-dependent kinase 5 (Cdk5) and its activator, p35, in cancer biology are explored one after another. We have previously demonstrated that Cdk5 may regulate proliferation of thyroid cancer cells. In addition, we also identify that Cdk5 overactivation can be triggered by drug treatments and leads to...

  4. Protein Kinase C Regulates the Cell Surface Activity of Endothelin-Converting Enzyme-1

    OpenAIRE

    Smith, A Ian; Lew, Rebecca A.; Thomas, Walter G; Tochon-Danguy, Nathalie

    2006-01-01

    The potent vasoconstrictor endothelin is a 21 amino acid peptide whose principal physiological function is to regulate vascular tone. The generation of endothelin is crucially dependent on the local presence and activity of endothelin converting enzyme-1 (ECE-1) expressed on the surface of vascular endothelial cells. In this study, we have shown in endothelial cells that the enzyme is phosphorylated, and that phosphorylation is increased by phorbol ester stimulation of protein kinase C (PKC)....

  5. The mucolipidosis IV Ca2+ channel TRPML1 (MCOLN1) is regulated by the TOR kinase.

    Science.gov (United States)

    Onyenwoke, Rob U; Sexton, Jonathan Z; Yan, Feng; Díaz, María Cristina Huertas; Forsberg, Lawrence J; Major, Michael B; Brenman, Jay E

    2015-09-15

    Autophagy is a complex pathway regulated by numerous signalling events that recycles macromolecules and may be perturbed in lysosomal storage disorders (LSDs). During autophagy, aberrant regulation of the lysosomal Ca(2+) efflux channel TRPML1 [transient receptor potential mucolipin 1 (MCOLN1)], also known as MCOLN1, is solely responsible for the human LSD mucolipidosis type IV (MLIV); however, the exact mechanisms involved in the development of the pathology of this LSD are unknown. In the present study, we provide evidence that the target of rapamycin (TOR), a nutrient-sensitive protein kinase that negatively regulates autophagy, directly targets and inactivates the TRPML1 channel and thereby functional autophagy, through phosphorylation. Further, mutating these phosphorylation sites to unphosphorylatable residues proved to block TOR regulation of the TRPML1 channel. These findings suggest a mechanism for how TOR activity may regulate the TRPML1 channel.

  6. Protein Kinase C Overactivity Impairs Prefrontal Cortical Regulation of Working Memory

    Science.gov (United States)

    Birnbaum, S. G.; Yuan, P. X.; Wang, M.; Vijayraghavan, S.; Bloom, A. K.; Davis, D. J.; Gobeske, K. T.; Sweatt, J. D.; Manji, H. K.; Arnsten, A. F. T.

    2004-10-01

    The prefrontal cortex is a higher brain region that regulates thought, behavior, and emotion using representational knowledge, operations often referred to as working memory. We tested the influence of protein kinase C (PKC) intracellular signaling on prefrontal cortical cognitive function and showed that high levels of PKC activity in prefrontal cortex, as seen for example during stress exposure, markedly impair behavioral and electrophysiological measures of working memory. These data suggest that excessive PKC activation can disrupt prefrontal cortical regulation of behavior and thought, possibly contributing to signs of prefrontal cortical dysfunction such as distractibility, impaired judgment, impulsivity, and thought disorder.

  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. Protein kinase D regulates RhoA activity via rhotekin phosphorylation.

    Science.gov (United States)

    Pusapati, Ganesh V; Eiseler, Tim; Rykx, An; Vandoninck, Sandy; Derua, Rita; Waelkens, Etienne; Van Lint, Johan; von Wichert, Götz; Seufferlein, Thomas

    2012-03-16

    The members of the protein kinase D (PKD) family of serine/threonine kinases are major targets for tumor-promoting phorbol esters, G protein-coupled receptors, and activated protein kinase C isoforms (PKCs). The expanding list of cellular processes in which PKDs exert their function via phosphorylation of various substrates include proliferation, apoptosis, migration, angiogenesis, and vesicle trafficking. Therefore, identification of novel PKD substrates is necessary to understand the profound role of this kinase family in signal transduction. Here, we show that rhotekin, an effector of RhoA GTPase, is a novel substrate of PKD. We identified Ser-435 in rhotekin as the potential site targeted by PKD in vivo. Expression of a phosphomimetic S435E rhotekin mutant resulted in an increase of endogenous active RhoA GTPase levels. Phosphorylation of rhotekin by PKD2 modulates the anchoring of the RhoA in the plasma membrane. Consequently, the S435E rhotekin mutant displayed enhanced stress fiber formation when expressed in serum-starved fibroblasts. Our data thus identify a novel role of PKD as a regulator of RhoA activity and actin stress fiber formation through phosphorylation of rhotekin. PMID:22228765

  9. Diverging regulation of pyruvate dehydrogenase kinase isoform gene expression in cultured human muscle cells.

    Science.gov (United States)

    Abbot, Emily L; McCormack, James G; Reynet, Christine; Hassall, David G; Buchan, Kevin W; Yeaman, Stephen J

    2005-06-01

    The pyruvate dehydrogenase complex occupies a central and strategic position in muscle intermediary metabolism and is primarily regulated by phosphorylation/dephosphorylation. The identification of multiple isoforms of pyruvate dehydrogenase kinase (PDK1-4) and pyruvate dehydrogenase phosphatase (PDP1-2) has raised intriguing new possibilities for chronic pyruvate dehydrogenase complex control. Experiments to date suggest that PDK4 is the major isoenzyme responsible for changes in pyruvate dehydrogenase complex activity in response to various different metabolic conditions. Using a cultured human skeletal muscle cell model system, we found that expression of both PDK2 and PDK4 mRNA is upregulated in response to glucose deprivation and fatty acid supplementation, the effects of which are reversed by insulin treatment. In addition, insulin directly downregulates PDK2 and PDK4 mRNA transcript abundance via a phosphatidylinositol 3-kinase-dependent pathway, which may involve glycogen synthase kinase-3 but does not utilize the mammalian target of rapamycin or mitogen-activated protein kinase signalling pathways. In order to further elucidate the regulation of PDK, the role of the peroxisome proliferators-activated receptors (PPAR) was investigated using highly potent subtype selective agonists. PPARalpha and PPARdelta agonists were found to specifically upregulate PDK4 mRNA expression, whereas PPARgamma activation selectively decreased PDK2 mRNA transcript abundance. PDP1 mRNA expression was unaffected by all conditions analysed. These results suggest that in human muscle, hormonal and nutritional conditions may control PDK2 and PDK4 mRNA expression via a common signalling mechanism. In addition, PPARs appear to independently regulate specific PDK isoform transcipt levels, which are likely to impart important metabolic mediation of fuel utilization by the muscle. PMID:15955060

  10. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

    Directory of Open Access Journals (Sweden)

    Lee A Borthwick

    Full Text Available Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR, a cAMP-dependent protein kinase A (PKA and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2 forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A. Overlay (Far-Western and Surface Plasmon Resonance (SPR analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727. Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.

  11. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

    Science.gov (United States)

    Borthwick, Lee A; Kerbiriou, Mathieu; Taylor, Christopher J; Cozza, Giorgio; Lascu, Ioan; Postel, Edith H; Cassidy, Diane; Trouvé, Pascal; Mehta, Anil; Robson, Louise; Muimo, Richmond

    2016-01-01

    Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.

  12. Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development.

    Science.gov (United States)

    Loponte, Sara; Segré, Chiara V; Senese, Silvia; Miccolo, Claudia; Santaguida, Stefano; Deflorian, Gianluca; Citro, Simona; Mattoscio, Domenico; Pisati, Federica; Moser, Mirjam A; Visintin, Rosella; Seiser, Christian; Chiocca, Susanna

    2016-01-01

    Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology. PMID:27458029

  13. The systematic functional analysis of plasmodium protein kinases identifies essential regulators of mosquito transmission

    KAUST Repository

    Tewari, Rita

    2010-10-21

    Although eukaryotic protein kinases (ePKs) contribute to many cellular processes, only three Plasmodium falciparum ePKs have thus far been identified as essential for parasite asexual blood stage development. To identify pathways essential for parasite transmission between their mammalian host and mosquito vector, we undertook a systematic functional analysis of ePKs in the genetically tractable rodent parasite Plasmodium berghei. Modeling domain signatures of conventional ePKs identified 66 putative Plasmodium ePKs. Kinomes are highly conserved between Plasmodium species. Using reverse genetics, we show that 23 ePKs are redundant for asexual erythrocytic parasite development in mice. Phenotyping mutants at four life cycle stages in Anopheles stephensi mosquitoes revealed functional clusters of kinases required for sexual development and sporogony. Roles for a putative SR protein kinase (SRPK) in microgamete formation, a conserved regulator of clathrin uncoating (GAK) in ookinete formation, and a likely regulator of energy metabolism (SNF1/KIN) in sporozoite development were identified. 2010 Elsevier Inc.

  14. Changes in dynamics upon oligomerization regulate substrate binding and allostery in amino acid kinase family members.

    Directory of Open Access Journals (Sweden)

    Enrique Marcos

    2011-09-01

    Full Text Available Oligomerization is a functional requirement for many proteins. The interfacial interactions and the overall packing geometry of the individual monomers are viewed as important determinants of the thermodynamic stability and allosteric regulation of oligomers. The present study focuses on the role of the interfacial interactions and overall contact topology in the dynamic features acquired in the oligomeric state. To this aim, the collective dynamics of enzymes belonging to the amino acid kinase family both in dimeric and hexameric forms are examined by means of an elastic network model, and the softest collective motions (i.e., lowest frequency or global modes of motions favored by the overall architecture are analyzed. Notably, the lowest-frequency modes accessible to the individual subunits in the absence of multimerization are conserved to a large extent in the oligomer, suggesting that the oligomer takes advantage of the intrinsic dynamics of the individual monomers. At the same time, oligomerization stiffens the interfacial regions of the monomers and confers new cooperative modes that exploit the rigid-body translational and rotational degrees of freedom of the intact monomers. The present study sheds light on the mechanism of cooperative inhibition of hexameric N-acetyl-L-glutamate kinase by arginine and on the allosteric regulation of UMP kinases. It also highlights the significance of the particular quaternary design in selectively determining the oligomer dynamics congruent with required ligand-binding and allosteric activities.

  15. Algal dual-specificity tyrosine phosphorylation-regulated kinase, triacylglycerol accumulation regulator1, regulates accumulation of triacylglycerol in nitrogen or sulfur deficiency.

    Science.gov (United States)

    Kajikawa, Masataka; Sawaragi, Yuri; Shinkawa, Haruka; Yamano, Takashi; Ando, Akira; Kato, Misako; Hirono, Masafumi; Sato, Naoki; Fukuzawa, Hideya

    2015-06-01

    Although microalgae accumulate triacylglycerol (TAG) and starch in response to nutrient-deficient conditions, the regulatory mechanisms are poorly understood. We report here the identification and characterization of a kinase, triacylglycerol accumulation regulator1 (TAR1), that is a member of the yeast (Saccharomyces cerevisiae) Yet another kinase1 (Yak1) subfamily in the dual-specificity tyrosine phosphorylation-regulated kinase family in a green alga (Chlamydomonas reinhardtii). The kinase domain of TAR1 showed auto- and transphosphorylation activities. A TAR1-defective mutant, tar1-1, accumulated TAG to levels 0.5- and 0.1-fold of those of a wild-type strain in sulfur (S)- and nitrogen (N)-deficient conditions, respectively. In N-deficient conditions, tar1-1 showed more pronounced arrest of cell division than the wild type, had increased cell size and cell dry weight, and maintained chlorophyll and photosynthetic activity, which were not observed in S-deficient conditions. In N-deficient conditions, global changes in expression levels of N deficiency-responsive genes in N assimilation and tetrapyrrole metabolism were noted between tar1-1 and wild-type cells. These results indicated that TAR1 is a regulator of TAG accumulation in S- and N-deficient conditions, and it functions in cell growth and repression of photosynthesis in conditions of N deficiency. PMID:25922058

  16. Regulation of ADAM12 cell-surface expression by protein kinase C epsilon

    DEFF Research Database (Denmark)

    Sundberg, Christina; Thodeti, Charles Kumar; Kveiborg, Marie;

    2004-01-01

    constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) epsilon induces ADAM12 translocation to the cell......The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembrane-anchored proteins are synthesized as proforms that have (from the N terminus): a prodomain; a metalloprotease-, disintegrin......-immunoprecipitated from membrane-enriched fractions of PMA-treated cells, 3) RD cells transfected with EGFP-tagged, myristoylated PKCepsilon expressed more ADAM12 at the cell surface than did non-transfected cells, and 4) RD cells transfected with a kinase-inactive PKCepsilon mutant did not exhibit ADAM12 cell...

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

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  18. Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

    Science.gov (United States)

    Abramovici, Hanan; Hogan, Angela B; Obagi, Christopher; Topham, Matthew K; Gee, Stephen H

    2003-11-01

    Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis. PMID:14551255

  19. Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

    Science.gov (United States)

    Abramovici, Hanan; Hogan, Angela B; Obagi, Christopher; Topham, Matthew K; Gee, Stephen H

    2003-11-01

    Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis.

  20. Eukaryotic elongation factor 2 kinase regulates the cold stress response by slowing translation elongation.

    Science.gov (United States)

    Knight, John R P; Bastide, Amandine; Roobol, Anne; Roobol, Jo; Jackson, Thomas J; Utami, Wahyu; Barrett, David A; Smales, C Mark; Willis, Anne E

    2015-01-15

    Cells respond to external stress conditions by controlling gene expression, a process which occurs rapidly via post-transcriptional regulation at the level of protein synthesis. Global control of translation is mediated by modification of translation factors to allow reprogramming of the translatome and synthesis of specific proteins that are required for stress protection or initiation of apoptosis. In the present study, we have investigated how global protein synthesis rates are regulated upon mild cooling. We demonstrate that although there are changes to the factors that control initiation, including phosphorylation of eukaryotic translation initiation factor 2 (eIF2) on the α-subunit, the reduction in the global translation rate is mediated by regulation of elongation via phosphorylation of eukaryotic elongation factor 2 (eEF2) by its specific kinase, eEF2K (eukaryotic elongation factor 2 kinase). The AMP/ATP ratio increases following cooling, consistent with a reduction in metabolic rates, giving rise to activation of AMPK (5'-AMP-activated protein kinase), which is upstream of eEF2K. However, our data show that the major trigger for activation of eEF2K upon mild cooling is the release of Ca2+ ions from the endoplasmic reticulum (ER) and, importantly, that it is possible to restore protein synthesis rates in cooled cells by inhibition of this pathway at multiple points. As cooling has both therapeutic and industrial applications, our data provide important new insights into how the cellular responses to this stress are regulated, opening up new possibilities to modulate these responses for medical or industrial use at physiological or cooler temperatures.

  1. Focal adhesion kinase regulates expression of thioredoxin-interacting protein (TXNIP) in cancer cells.

    Science.gov (United States)

    Ho, Baotran; Huang, Grace; Golubovskaya, Vita M

    2014-01-01

    Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter activity. Treatment of MCF-7 cells with 1alpha, 25-dihydroxyvitamin D3 (1,25D) down-regulated endogenous FAK and up-regulated TXNIP protein level, and treatment with 5-FU decreased FAK protein expression and up-regulated TXNIP protein expression in 293 cells. Moreover, silencing of FAK with siRNA increased TXNIP protein expression, while overexpression of FAK inhibited TXNIP protein expression in 293 cells. In addition, treatment of DBTRG glioblastoma cells with FAK inhibitor Y15 increased TXNIP mRNA, decreased cancer cell viability and increased apoptosis. These results for the first time demonstrate FAK-regulated TXNIP expression which is important for apoptotic, survival and oxidative stress signaling pathways in cancer cells. PMID:23387972

  2. Regulation of extracellular signal-regulated kinase 1/2 inlfuences hippocampal neuronal survival in a rat model of diabetic cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Yaning Zhao; Jianmin Li; Qiqun Tang; Pan Zhang; Liwei Jing; Changxiang Chen; Shuxing Li

    2014-01-01

    Activation of extracellular signal-regulated kinase 1/2 has been demonstrated in acute brain ischemia. We hypothesized that activated extracellular signal-regulated kinase 1/2 can protect hippocampal neurons from injury in a diabetic model after cerebral ischemia/reperfusion. In this study, transient whole-brain ischemia was induced by four-vessel occlusion in normal and diabetic rats, and extracellular signal-regulated kinase 1/2 inhibitor (U0126) was administered into diabetic rats 30 minutes before ischemia as a pretreatment. Results showed that the number of surviving neurons in the hippocampal CA1 region was reduced, extracellular signal-regulated kinase 1/2 phosphorylation and Ku70 activity were decreased, and pro-apoptotic Bax expression was upregulated after intervention using U0126. These ifndings demonstrate that inhibition of extracellular signal-regulated kinase 1/2 activity aggravated neuronal loss in the hippocampus in a diabetic rat after cerebral ischemia/reperfusion, further decreased DNA repairing ability and ac-celerated apoptosis in hippocampal neurons. Extracellular signal-regulated kinase 1/2 activation plays a neuroprotective role in hippocampal neurons in a diabetic rat after cerebral ischemia/reperfusion.

  3. Effects of Arg-Gly-Asp-modified elastin-like polypeptide on pseudoislet formation via up-regulation of cell adhesion molecules and extracellular matrix proteins.

    Science.gov (United States)

    Lee, Kyeong-Min; Jung, Gwon-Soo; Park, Jin-Kyu; Choi, Seong-Kyoon; Jeon, Won Bae

    2013-03-01

    Extracellular matrix (ECM) plays an important role in controlling the β-cell morphology, survival and insulin secretary functions. An RGD-modified elastin-like polypeptide (RGD-ELP), TGPG[VGRGD(VGVPG)(6)](20)WPC, has been reported previously as a bioactive matrix. In this study, to investigate whether RGD-ELP affects β-cell growth characteristics and insulin secretion, β-TC6 cells were cultured on the RGD-ELP coatings prepared via thermally induced phase transition. On RGD-ELP, β-TC6 cells clustered into an islet-like architecture with high cell viability. Throughout 7days' culture, the proliferation rate of the cells within a pseudoislet was similar to that of monolayer culture. Under high glucose (25mM), β-TC6 pseudoislets showed up-regulated insulin gene expression and exhibited glucose-stimulated insulin secretion. Importantly, the mRNA and protein abundances of cell adhesion molecules (CAM) E-cadherin and connexin-36 were much higher in pseudoislets than in monolayer cells. The siRNA-mediated inhibition of E-cadherin or connexin-36 expression severely limited pseudoislet formation. In addition, the mRNA levels of collagen types I and IV, fibronectin and laminin were significantly elevated in pseudoislets. The results suggest that RGD-ELP promotes pseudoislet formation via up-regulation of the CAM and ECM components. The functional roles of RGD-ELP are discussed in respect of its molecular composition.

  4. Regulation of pyruvate dehydrogenase kinase expression by the farnesoid X receptor

    International Nuclear Information System (INIS)

    The pyruvate dehydrogenase complex (PDC) functions as an important junction in intermediary metabolism by influencing the utilization of fat versus carbohydrate as a source of fuel. Activation of PDC is achieved by phosphatases, whereas, inactivation is catalyzed by pyruvate dehydrogenase kinases (PDKs). The expression of PDK4 is highly regulated by the glucocorticoid and peroxisome proliferator-activated receptors. We demonstrate that the farnesoid X receptor (FXR; NR1H4), which regulates a variety of genes involved in lipoprotein metabolism, also regulates the expression of PDK4. Treatment of rat hepatoma cells as well as human primary hepatocytes with FXR agonists stimulates the expression of PDK4 to levels comparable to those obtained with glucocorticoids. In addition, treatment of mice with an FXR agonist significantly increased hepatic PDK4 expression, while concomitantly decreasing plasma triglyceride levels. Thus, activation of FXR may suppress glycolysis and enhance oxidation of fatty acids via inactivation of the PDC by increasing PDK4 expression

  5. Circadian and pharmacological regulation of casein kinase I in the hamster suprachiasmatic nucleus

    Indian Academy of Sciences (India)

    Patricia V. Agostino; Santiago A. Plano; Diego A. Golombek

    2008-12-01

    In mammals, the mechanism for the generation of circadian rhythms and entrainment by light–dark (LD) cycles resides in the hypothalamic suprachiasmatic nuclei (SCN), and the principal signal that adjusts this biological clock with environmental timing is the light:dark cycle. Within the SCN, rhythms are generated by a complex of molecular feedback loops that regulate the transcription of clock genes, including per and cry. Posttranslational modification plays an essential role in the regulation of biological rhythms; in particular, clock gene phosphorylation by casein kinase I, both epsilon (CKI) and delta (CKI), regulates key molecular mechanisms in the circadian clock. In this paper, we report for the first time that CKI activity undergoes a significant circadian rhythm in the SCN (peaking at circadian time 12, the start of the subjective night), and its pharmacological inhibition alters photic entrainment of the clock, indicating that CKI may be a key element in this pathway.

  6. Phosphatidylinositol 4-phosphate 5-kinases in the regulation of T cell activation

    Directory of Open Access Journals (Sweden)

    Loretta eTuosto

    2016-05-01

    Full Text Available Phosphatidylinositol 4,5-biphosphate kinases (PIP5K are critical regulators of T cell activation being the main enzymes involved in the synthesis of phosphatidylinositol 4,5-biphosphate (PIP2. PIP2 is indeed a pivotal regulator of the actin cytoskeleton, thus controlling T cell polarization and migration, stable adhesion to antigen presenting cells (APC, spatial organization of the immunological synapse (IS, and costimulation. Moreover, PIP2 serves also as a precursor for the second messengers inositol triphosphate (IP3, diacylglycerol (DAG and phosphatidylinositol 3,4,5-triphosphate (PIP3, which are essential for the activation of signalling pathways regulating cytokine production, cell cycle progression, survival, metabolism and differentiation. Here, we discuss the impact of PIP5Ks on several T lymphocyte functions with a specific focus on the role of CD28 co-stimulation in PIP5K compartimentalization and activation.

  7. Identification of a site critical for kinase regulation on the central processing unit (CPU) helix of the aspartate receptor.

    Science.gov (United States)

    Trammell, M A; Falke, J J

    1999-01-01

    Ligand binding to the homodimeric aspartate receptor of Escherichia coli and Salmonella typhimurium generates a transmembrane signal that regulates the activity of a cytoplasmic histidine kinase, thereby controlling cellular chemotaxis. This receptor also senses intracellular pH and ambient temperature and is covalently modified by an adaptation system. A specific helix in the cytoplasmic domain of the receptor, helix alpha6, has been previously implicated in the processing of these multiple input signals. While the solvent-exposed face of helix alpha6 possesses adaptive methylation sites known to play a role in kinase regulation, the functional significance of its buried face is less clear. This buried region lies at the subunit interface where helix alpha6 packs against its symmetric partner, helix alpha6'. To test the role of the helix alpha6-helix alpha6' interface in kinase regulation, the present study introduces a series of 13 side-chain substitutions at the Gly 278 position on the buried face of helix alpha6. The substitutions are observed to dramatically alter receptor function in vivo and in vitro, yielding effects ranging from kinase superactivation (11 examples) to complete kinase inhibition (one example). Moreover, four hydrophobic, branched side chains (Val, Ile, Phe, and Trp) lock the kinase in the superactivated state regardless of whether the receptor is occupied by ligand. The observation that most side-chain substitutions at position 278 yield kinase superactivation, combined with evidence that such facile superactivation is rare at other receptor positions, identifies the buried Gly 278 residue as a regulatory hotspot where helix packing is tightly coupled to kinase regulation. Together, helix alpha6 and its packing interactions function as a simple central processing unit (CPU) that senses multiple input signals, integrates these signals, and transmits the output to the signaling subdomain where the histidine kinase is bound. Analogous CPU

  8. Regulation of vascular endothelial growth factor expression by homeodomain-interacting protein kinase-2

    Directory of Open Access Journals (Sweden)

    D'Orazi Gabriella

    2008-07-01

    Full Text Available Abstract Background Homeodomain-interacting protein kinase-2 (HIPK2 plays an essential role in restraining tumor progression as it may regulate, by itself or within multiprotein complexes, many proteins (mainly transcription factors involved in cell growth and apoptosis. This study takes advantage of the recent finding that HIPK2 may repress the β-catenin transcription activity. Thus, we investigated whether HIPK2 overexpression may down-regulate vascular endothelial growth factor (VEGF levels (a β-catenin target gene and the role of β-catenin in this regulation, in order to consider HIPK2 as a tool for novel anti-tumoral therapeutical approaches. Methods The regulation of VEGF expression by HIPK2 was evaluated by using luciferase assay with VEGF reporter construct, after overexpression of the β-catenin transcription factor. Relative quantification of VEGF and β-catenin mRNAs were assessed by reverse-transcriptase-PCR (RT-PCR analyses, following HIPK2 overexpression, while β-catenin protein levels were evaluated by western immunoblotting. Results HIPK2 overexpression in tumor cells downregulated VEGF mRNA levels and VEGF promoter activity. The VEGF downregulation was partly depending on HIPK2-mediated β-catenin regulation. Thus, HIPK2 could induce β-catenin protein degradation that was prevented by cell treatment with proteasome inhibitor MG132. The β-catenin degradation was dependent on HIPK2 catalytic activity and independent of p53 and glycogen synthase kinase 3β (GSK-3β activities. Conclusion These results suggest that VEGF might be a target of HIPK2, at least in part, through regulation of β-catenin activity. These findings support the function of HIPK2 as tumor suppressor and hypothesise a role for HIPK2 as antiangiogenic tool in tumor therapy approaches.

  9. Akt Inhibitor A-443654 Interferes with Mitotic Progression by Regulating Aurora A Kinase Expression

    Directory of Open Access Journals (Sweden)

    Xuesong Liu

    2008-08-01

    Full Text Available Both Akt and Aurora A kinase have been shown to be important targets for intervention for cancer therapy. We report here that Compound A (A-443654, a specific Akt inhibitor, interferes with mitotic progression and bipolar spindle formation. Compound A induces G2/M accumulation, defects in centrosome separation, and formation of either monopolar arrays or disorganized spindles. On the basis of gene expression array studies, we identified Aurora A as one of the genes regulated transcriptionally by Akt inhibitors including Compound A. Inhibition of the phosphatidylinositol 3-kinase (PI3K/Akt pathway, either by PI3K inhibitor LY294002 or by Compound A, dramatically inhibits the promoter activity of Aurora A, whereas the mammalian target of rapamycin inhibitor has little effect, suggesting that Akt might be responsible for up-regulating Aurora A for mitotic progression. Further analysis of the Aurora A promoter region indicates that the Ets element but not the Sp1 element is required for Compound A-sensitive transcriptional control of Aurora A. Overexpression of Aurora A in cells treated with Compound A attenuates the mitotic arrest and the defects in bipolar spindle formation induced by Akt inhibition. Our studies suggest that that Akt may promote mitotic progression through the transcriptional regulation of Aurora A.

  10. Regulation of Renal Electrolyte Transport by WNK and SPAK-OSR1 Kinases.

    Science.gov (United States)

    Hadchouel, Juliette; Ellison, David H; Gamba, Gerardo

    2016-01-01

    The discovery of four genes responsible for pseudohypoaldosteronism type II, or familial hyperkalemic hypertension, which features arterial hypertension with hyperkalemia and metabolic acidosis, unmasked a complex multiprotein system that regulates electrolyte transport in the distal nephron. Two of these genes encode the serine-threonine kinases WNK1 and WNK4. The other two genes [kelch-like 3 (KLHL3) and cullin 3 (CUL3)] form a RING-type E3-ubiquitin ligase complex that modulates WNK1 and WNK4 abundance. WNKs regulate the activity of the Na(+):Cl(-) cotransporter (NCC), the epithelial sodium channel (ENaC), the renal outer medullary potassium channel (ROMK), and other transport pathways. Interestingly, the modulation of NCC occurs via the phosphorylation by WNKs of other serine-threonine kinases known as SPAK-OSR1. In contrast, the process of regulating the channels is independent of SPAK-OSR1. We present a review of the remarkable advances in this area in the past 10 years. PMID:26863326

  11. Regulation of Ikaros function by casein kinase 2 and protein phosphatase 1

    Institute of Scientific and Technical Information of China (English)

    Amy; K; Erbe; Aleksandar; Savic; Sinisa; Dovat

    2011-01-01

    The Ikaros gene encodes a zinc finger,DNA-binding protein that regulates gene transcription and chromatin remodeling.Ikaros is a master regulator of hematopoiesis and an established tumor suppressor.Moderate alteration of Ikaros activity (e.g.haploinsufficiency) appears to be sufficient to promote malignant transformation in human hematopoietic cells.This raises questions about the mechanisms that normally regulate Ikaros function and the potential of these mechanisms to contribute to the development of leukemia.The focus of this review is the regulation of Ikaros function by phosphorylation/dephosphorylation.Site-specific phosphorylation of Ikaros by casein kinase 2 (CK2) controls Ikaros DNA-binding ability and subcellular localization.As a consequence,the ability of Ikaros to regulate cell cycle progression,chromatin remodeling,target gene expression,and thymocyte differentiation are controlled by CK2.In addition,hyperphosphorylation of Ikaros by CK2 leads to decreased Ikaros levels due to ubiquitinmediated degradation.Dephosphorylation of Ikaros by protein phosphatase 1 (PP1) acts in opposition to CK2 to increase Ikaros stability and restore Ikaros DNA binding ability and pericentromeric localization.Thus,the CK2 and PP1 pathways act in concert to regulate Ikaros activity in hematopoiesis and as a tumor suppressor.This highlights the importance of these signal transduction pathways as potential mediators of leukemogenesis via their role in regulating the activities of Ikaros.

  12. The AMPK-related kinase SNARK regulates muscle mass and myocyte survival

    Science.gov (United States)

    Lessard, Sarah J.; Rivas, Donato A.; So, Kawai; Koh, Ho-Jin; Queiroz, André Lima; Hirshman, Michael F.; Fielding, Roger A.; Goodyear, Laurie J.

    2015-01-01

    The maintenance of skeletal muscle mass is critical for sustaining health; however, the mechanisms responsible for muscle loss with aging and chronic diseases, such as diabetes and obesity, are poorly understood. We found that expression of a member of the AMPK-related kinase family, the SNF1-AMPK-related kinase (SNARK, also known as NUAK2), increased with muscle cell differentiation. SNARK expression increased in skeletal muscles from young mice exposed to metabolic stress and in muscles from healthy older human subjects. The regulation of SNARK expression in muscle with differentiation and physiological stress suggests that SNARK may function in the maintenance of muscle mass. Consistent with this hypothesis, decreased endogenous SNARK expression (using siRNA) in cultured muscle cells resulted in increased apoptosis and decreased cell survival under conditions of metabolic stress. Likewise, muscle-specific transgenic animals expressing a SNARK dominant-negative inactive mutant (SDN) had increased myonuclear apoptosis and activation of apoptotic mediators in muscle. Moreover, animals expressing SDN had severe, age-accelerated muscle atrophy and increased adiposity, consistent with sarcopenic obesity. Reduced SNARK activity, in vivo and in vitro, caused downregulation of the Rho kinase signaling pathway, a key mediator of cell survival. These findings reveal a critical role for SNARK in myocyte survival and the maintenance of muscle mass with age. PMID:26690705

  13. The AMPK-related kinase SNARK regulates muscle mass and myocyte survival.

    Science.gov (United States)

    Lessard, Sarah J; Rivas, Donato A; So, Kawai; Koh, Ho-Jin; Queiroz, André Lima; Hirshman, Michael F; Fielding, Roger A; Goodyear, Laurie J

    2016-02-01

    The maintenance of skeletal muscle mass is critical for sustaining health; however, the mechanisms responsible for muscle loss with aging and chronic diseases, such as diabetes and obesity, are poorly understood. We found that expression of a member of the AMPK-related kinase family, the SNF1-AMPK-related kinase (SNARK, also known as NUAK2), increased with muscle cell differentiation. SNARK expression increased in skeletal muscles from young mice exposed to metabolic stress and in muscles from healthy older human subjects. The regulation of SNARK expression in muscle with differentiation and physiological stress suggests that SNARK may function in the maintenance of muscle mass. Consistent with this hypothesis, decreased endogenous SNARK expression (using siRNA) in cultured muscle cells resulted in increased apoptosis and decreased cell survival under conditions of metabolic stress. Likewise, muscle-specific transgenic animals expressing a SNARK dominant-negative inactive mutant (SDN) had increased myonuclear apoptosis and activation of apoptotic mediators in muscle. Moreover, animals expressing SDN had severe, age-accelerated muscle atrophy and increased adiposity, consistent with sarcopenic obesity. Reduced SNARK activity, in vivo and in vitro, caused downregulation of the Rho kinase signaling pathway, a key mediator of cell survival. These findings reveal a critical role for SNARK in myocyte survival and the maintenance of muscle mass with age. PMID:26690705

  14. Protein kinase C mediates cholinergically regulated protein phosphorylation in a Cl(-)-secreting epithelium.

    Science.gov (United States)

    Cohn, J A

    1990-02-01

    T84 cell monolayers were used to study the cholinergic regulation of protein phosphorylation in epithelial cells. When T84 cell monolayers are labeled with 32Pi and stimulated with carbachol, six proteins exhibit altered phosphorylation. The most prominent response is a fivefold increase in labeling of p83, an acidic protein of Mr 83,000. Increasing labeling of p83 parallels stimulated secretion with respect to the onset of agonist action, agonist potency, and antagonism by atropine. However, the p83 and secretory responses differ in that the p83 response is more sustained. When T84 cell fractions are incubated with [gamma-32P]ATP, Ca2(+)-phospholipid stimulates p83 labeling. Phosphorylation of p83 also occurs when a T84 cell extract is incubated with purified protein kinase C and when intact cells are exposed to phorbol myristate acetate. p83 does not become phosphorylated in cell fractions incubated with adenosine 3',5'-cyclic monophosphate (cAMP) or in monolayers stimulated with agonists acting via cAMP. Thus carbachol stimulates the phosphorylation of an endogenous substrate for protein kinase C in T84 cells. The duration of this phosphorylation response suggests that protein kinase C may mediate a sustained response to carbachol, possibly acting to limit the duration of stimulated secretion.

  15. Exploitation of latent protein allostery enables the evolution of novel and divergent MAP kinase regulation

    Science.gov (United States)

    Coyle, Scott M.; Flores, Jonathan; Lim, Wendell A.

    2013-01-01

    SUMMARY Allosteric interactions provide precise spatiotemporal control over signaling proteins, but how allosteric activators and their targets co-evolve is poorly understood. Here, we trace the evolution of two allosteric activator motifs within the yeast scaffold protein Ste5 that specifically target the mating MAP kinase Fus3. One activator (Ste5-VWA) provides pathway insulation and dates to the divergence of Fus3 from its paralog, Kss1; a second activator (Ste5-FBD) that tunes mating behavior is, in contrast, not conserved in most lineages. Surprisingly, both Ste5 activator motifs could regulate MAP kinases that diverged from Fus3 prior to the emergence of Ste5, suggesting that Ste5 activators arose by exploiting latent regulatory features already present in the MAPK ancestor. The magnitude of this latent allosteric potential drifts widely among pre-Ste5 MAP kinases, providing a pool of hidden phenotypic diversity that, when revealed by new activators, could lead to functional divergence and the evolution of distinct signaling behaviors. PMID:23953117

  16. Exploitation of latent allostery enables the evolution of new modes of MAP kinase regulation.

    Science.gov (United States)

    Coyle, Scott M; Flores, Jonathan; Lim, Wendell A

    2013-08-15

    Allosteric interactions provide precise spatiotemporal control over signaling proteins, but how allosteric activators and their targets coevolve is poorly understood. Here, we trace the evolution of two allosteric activator motifs within the yeast scaffold protein Ste5 that specifically target the mating MAP kinase Fus3. One activator (Ste5-VWA) provides pathway insulation and dates to the divergence of Fus3 from its paralog, Kss1; a second activator (Ste5-FBD) that tunes mating behavior is, in contrast, not conserved in most lineages. Surprisingly, both Ste5 activator motifs could regulate MAP kinases that diverged from Fus3 prior to the emergence of Ste5, suggesting that Ste5 activators arose by exploiting latent regulatory features already present in the MAPK ancestor. The magnitude of this latent allosteric potential drifts widely among pre-Ste5 MAP kinases, providing a pool of hidden phenotypic diversity that, when revealed by new activators, could lead to functional divergence and to the evolution of distinct signaling behaviors. PMID:23953117

  17. AMP-activated protein kinase regulates nicotinamide phosphoribosyl transferase expression in skeletal muscle

    DEFF Research Database (Denmark)

    Brandauer, Josef; Vienberg, Sara Gry; Andersen, Marianne Agerholm;

    2013-01-01

    -activated protein kinase (AMPK) increases sirtuin activity by elevating NAD levels. As NAM directly inhibits sirtuins, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary for...... increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training- or 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR)-mediated increases in skeletal muscle Nampt abundance are AMPK dependant. One-legged knee-extensor exercise training in humans increased Nampt protein...

  18. M2 pyruvate kinase provides a mechanism for nutrient sensing and regulation of cell proliferation

    OpenAIRE

    Morgan, Hugh P.; O???Reilly, Francis J.; Wear, Martin A.; O'Neill, Robert; Fothergill-Gilmore, Linda A.; Hupp, Ted; Walkinshaw, Malcolm D.

    2013-01-01

    We show that the M2 isoform of pyruvate kinase (M2PYK) exists in equilibrium between monomers and tetramers regulated by allosteric binding of naturally occurring small-molecule metabolites. Phenylalanine stabilizes an inactive T-state tetrameric conformer and inhibits M2PYK with an IC50 value of 0.24 mM, whereas thyroid hormone (triiodo-l-thyronine, T3) stabilizes an inactive monomeric form of M2PYK with an IC50 of 78 nM. The allosteric activator fructose-1,6-bisphosphate [F16BP, AC50 (conce...

  19. Ethanol Regulation of Synaptic GABAA α4 Receptors Is Prevented by Protein Kinase A Activation.

    Science.gov (United States)

    Carlson, Stephen L; Bohnsack, John Peyton; Morrow, A Leslie

    2016-04-01

    Ethanol alters GABAA receptor trafficking and function through activation of protein kinases, and these changes may underlie ethanol dependence and withdrawal. In this study, we used subsynaptic fraction techniques and patch-clamp electrophysiology to investigate the biochemical and functional effects of protein kinase A (PKA) and protein kinase C (PKC) activation by ethanol on synaptic GABAA α4 receptors, a key target of ethanol-induced changes. Rat cerebral cortical neurons were grown for 18 days in vitro and exposed to ethanol and/or kinase modulators for 4 hours, a paradigm that recapitulates GABAergic changes found after chronic ethanol exposure in vivo. PKA activation by forskolin or rolipram during ethanol exposure prevented increases in P2 fraction α4 subunit abundance, whereas inhibiting PKA had no effect. Similarly, in the synaptic fraction, activation of PKA by rolipram in the presence of ethanol prevented the increase in synaptic α4 subunit abundance, whereas inhibiting PKA in the presence of ethanol was ineffective. Conversely, PKC inhibition in the presence of ethanol prevented the ethanol-induced increases in synaptic α4 subunit abundance. Finally, we found that either activating PKA or inhibiting PKC in the presence of ethanol prevented the ethanol-induced decrease in GABA miniature inhibitory postsynaptic current decay τ1, whereas inhibiting PKA had no effect. We conclude that PKA and PKC have opposing effects in the regulation of synaptic α4 receptors, with PKA activation negatively modulating, and PKC activation positively modulating, synaptic α4 subunit abundance and function. These results suggest potential targets for restoring normal GABAergic functioning in the treatment of alcohol use disorders.

  20. LIK1, a CERK1-interacting kinase, regulates plant immune responses in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Mi Ha Le

    Full Text Available Chitin, an integral component of the fungal cell wall, is one of the best-studied microbe-associated molecular patterns. Previous work identified a LysM receptor-like kinase (LysM-RLK1/CERK1 as the primary chitin receptor in Arabidopsis. In order to identify proteins that interact with CERK1, we conducted a yeast two-hybrid screen using the intracellular kinase domain of CERK1 as the bait. This screen identified 54 putative CERK1-interactors. Screening mutants defective in 43 of these interacting proteins identified only two, a calmodulin like protein (At3g10190 and a leucine-rich repeat receptor like kinase (At3g14840, which differed in their response to pathogen challenge. In the present work, we focused on characterizing the LRR-RLK gene where mutations altered responses to chitin elicitation. This LRR-RLK was named LysM RLK1-interacting kinase 1 (LIK1. The interaction between CERK1 and LIK1 was confirmed by co-immunoprecipitation using protoplasts and transgenic plants. In vitro experiments showed that LIK1 was directly phosphorylated by CERK1. In vivo phosphorylation assays showed that Col-0 wild-type plants have more phosphorylated LIK1 than cerk1 mutant plants, suggesting that LIK1 may be directly phosphorylated by CERK1. Lik1 mutant plants showed an enhanced response to both chitin and flagellin elicitors. In comparison to the wild-type plants, lik1 mutant plants were more resistant to the hemibiotrophic pathogen Pseudomonas syringae, but more susceptible to the necrotrophic pathogen Sclerotinia sclerotiorum. Consistent with the enhanced susceptibility to necrotrophs, lik1 mutants showed reduced expression of genes involved in jasmonic acid and ethylene signaling pathways. These data suggest that LIK1 directly interacts with CERK1 and regulates MAMP-triggered innate immunity.

  1. Negative regulation of active zone assembly by a newly identified SR protein kinase.

    Directory of Open Access Journals (Sweden)

    Ervin L Johnson

    2009-09-01

    Full Text Available Presynaptic, electron-dense, cytoplasmic protrusions such as the T-bar (Drosophila or ribbon (vertebrates are believed to facilitate vesicle movement to the active zone (AZ of synapses throughout the nervous system. The molecular composition of these structures including the T-bar and ribbon are largely unknown, as are the mechanisms that specify their synapse-specific assembly and distribution. In a large-scale, forward genetic screen, we have identified a mutation termed air traffic controller (atc that causes T-bar-like protein aggregates to form abnormally in motoneuron axons. This mutation disrupts a gene that encodes for a serine-arginine protein kinase (SRPK79D. This mutant phenotype is specific to SRPK79D and is not secondary to impaired kinesin-dependent axonal transport. The srpk79D gene is neuronally expressed, and transgenic rescue experiments are consistent with SRPK79D kinase activity being necessary in neurons. The SRPK79D protein colocalizes with the T-bar-associated protein Bruchpilot (Brp in both the axon and synapse. We propose that SRPK79D is a novel T-bar-associated protein kinase that represses T-bar assembly in peripheral axons, and that SRPK79D-dependent repression must be relieved to facilitate site-specific AZ assembly. Consistent with this model, overexpression of SRPK79D disrupts AZ-specific Brp organization and significantly impairs presynaptic neurotransmitter release. These data identify a novel AZ-associated protein kinase and reveal a new mechanism of negative regulation involved in AZ assembly. This mechanism could contribute to the speed and specificity with which AZs are assembled throughout the nervous system.

  2. 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

  3. A conserved dimorphism-regulating histidine kinase controls the dimorphic switching in Paracoccidioides brasiliensis.

    Science.gov (United States)

    Chaves, Alison F A; Navarro, Marina V; Castilho, Daniele G; Calado, Juliana C P; Conceição, Palloma M; Batista, Wagner L

    2016-08-01

    Paracoccidioides brasiliensis and P. lutzii, thermally dimorphic fungi, are the causative agents of paracoccidioidomycosis (PCM). Paracoccidioides infection occurs when conidia or mycelium fragments are inhaled by the host, which causes the Paracoccidioides cells to transition to the yeast form. The development of disease requires conidia inside the host alveoli to differentiate into yeast cells in a temperature-dependent manner. We describe the presence of a two-component signal transduction system in P. brasiliensis, which we investigated by expression analysis of a hypothetical protein gene (PADG_07579) that showed high similarity with the dimorphism-regulating histidine kinase (DRK1) gene of Blastomyces dermatitidis and Histoplasma capsulatum This gene was sensitive to environmental redox changes, which was demonstrated by a dose-dependent decrease in transcript levels after peroxide stimulation and a subtler decrease in transcript levels after NO stimulation. Furthermore, the higher PbDRK1 levels after treatment with increasing NaCl concentrations suggest that this histidine kinase can play a role as osmosensing. In the mycelium-yeast (M→Y) transition, PbDRK1 mRNA expression increased 14-fold after 24 h incubation at 37°C, consistent with similar observations in other virulent fungi. These results demonstrate that the PbDRK1 gene is differentially expressed during the dimorphic M→Y transition. Finally, when P. brasiliensis mycelium cells were exposed to a histidine kinase inhibitor and incubated at 37°C, there was a delay in the dimorphic M→Y transition, suggesting that histidine kinases could be targets of interest for PCM therapy. PMID:27268997

  4. Regulation of Ribosomal S6 Protein Kinase-p90rsk, Glycogen Synthase Kinase 3, and β-Catenin in Early Xenopus Development

    OpenAIRE

    Torres, Monica A.; Eldar-Finkelman, Hagit; Krebs, Edwin G.; Moon, Randall T.

    1999-01-01

    β-Catenin is a multifunctional protein that binds cadherins at the plasma membrane, HMG box transcription factors in the nucleus, and several cytoplasmic proteins that are involved in regulating its stability. In developing embryos and in some human cancers, the accumulation of β-catenin in the cytoplasm and subsequently the nuclei of cells may be regulated by the Wnt-1 signaling cascade and by glycogen synthase kinase 3 (GSK-3). This has increased interest in regulators of both GSK-3 and β-c...

  5. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of); Shin, Incheol, E-mail: incheol@hanyang.ac.kr [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of)

    2013-02-01

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  6. Identifying a kinase network regulating FGF14:Nav1.6 complex assembly using split-luciferase complementation.

    Directory of Open Access Journals (Sweden)

    Wei-Chun Hsu

    Full Text Available Kinases play fundamental roles in the brain. Through complex signaling pathways, kinases regulate the strength of protein:protein interactions (PPI influencing cell cycle, signal transduction, and electrical activity of neurons. Changes induced by kinases on neuronal excitability, synaptic plasticity and brain connectivity are linked to complex brain disorders, but the molecular mechanisms underlying these cellular events remain for the most part elusive. To further our understanding of brain disease, new methods for rapidly surveying kinase pathways in the cellular context are needed. The bioluminescence-based luciferase complementation assay (LCA is a powerful, versatile toolkit for the exploration of PPI. LCA relies on the complementation of two firefly luciferase protein fragments that are functionally reconstituted into the full luciferase enzyme by two interacting binding partners. Here, we applied LCA in live cells to assay 12 kinase pathways as regulators of the PPI complex formed by the voltage-gated sodium channel, Nav1.6, a transmembrane ion channel that elicits the action potential in neurons and mediates synaptic transmission, and its multivalent accessory protein, the fibroblast growth factor 14 (FGF14. Through extensive dose-dependent validations of structurally-diverse kinase inhibitors and hierarchical clustering, we identified the PI3K/Akt pathway, the cell-cycle regulator Wee1 kinase, and protein kinase C (PKC as prospective regulatory nodes of neuronal excitability through modulation of the FGF14:Nav1.6 complex. Ingenuity Pathway Analysis shows convergence of these pathways on glycogen synthase kinase 3 (GSK3 and functional assays demonstrate that inhibition of GSK3 impairs excitability of hippocampal neurons. This combined approach provides a versatile toolkit for rapidly surveying PPI signaling, allowing the discovery of new modular pathways centered on GSK3 that might be the basis for functional alterations between the

  7. 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.

  8. Bacillus subtilis Two-Component System Sensory Kinase DegS Is Regulated by Serine Phosphorylation in Its Input Domain

    DEFF Research Database (Denmark)

    Jers, Carsten; Kobir, Ahasanul; Søndergaard, Elsebeth Oline;

    2011-01-01

    Bacillus subtilis two-component system DegS/U is well known for the complexity of its regulation. The cytosolic sensory kinase DegS does not receive a single predominant input signal like most two-component kinases, instead it integrates a wide array of metabolic inputs that modulate its activity...... demonstrate that DegS phosphorylation can be carried out by at least two B. subtilis Hanks-type kinases in vitro, and this stimulates the phosphate transfer towards DegU. The consequences of this process were studied in vivo, using phosphomimetic (Ser76Asp) and non-phosphorylatable (Ser76Ala) mutants of Deg...

  9. The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis

    DEFF Research Database (Denmark)

    Yde, C W; Olsen, B B; Meek, D;

    2008-01-01

    Cell-cycle transition from the G(2) phase into mitosis is regulated by the cyclin-dependent protein kinase 1 (CDK1) in complex with cyclin B. CDK1 activity is controlled by both inhibitory phosphorylation, catalysed by the Myt1 and Wee1 kinases, and activating dephosphorylation, mediated by the CDC......25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory beta-subunit of protein kinase CK2 by RNA...

  10. 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....

  11. AMP-activated protein kinase in contraction regulation of skeletal muscle metabolism: necessary and/or sufficient?

    DEFF Research Database (Denmark)

    Jensen, Thomas Elbenhardt; Wojtaszewski, Jørgen; Richter, Erik

    2009-01-01

    In skeletal muscle, the contraction-activated heterotrimeric 5'-AMP-activated protein kinase (AMPK) protein is proposed to regulate the balance between anabolic and catabolic processes by increasing substrate uptake and turnover in addition to regulating the transcription of proteins involved...

  12. Spatial regulation of the cAMP-dependent protein kinase during chemotactic cell migration.

    Science.gov (United States)

    Howe, Alan K; Baldor, Linda C; Hogan, Brian P

    2005-10-01

    Historically, the cAMP-dependent protein kinase (PKA) has a paradoxical role in cell motility, having been shown to both facilitate and inhibit actin cytoskeletal dynamics and cell migration. In an effort to understand this dichotomy, we show here that PKA is regulated in subcellular space during cell migration. Immunofluorescence microscopy and biochemical enrichment of pseudopodia showed that type II regulatory subunits of PKA and PKA activity are enriched in protrusive cellular structures formed during chemotaxis. This enrichment correlates with increased phosphorylation of key cytoskeletal substrates for PKA, including the vasodilator-stimulated phosphoprotein (VASP) and the protein tyrosine phosphatase containing a PEST motif. Importantly, inhibition of PKA activity or its ability to interact with A kinase anchoring proteins inhibited the activity of the Rac GTPase within pseudopodia. This effect correlated with both decreased guanine nucleotide exchange factor activity and increased GTPase activating protein activity. Finally, inhibition of PKA anchoring, like inhibition of total PKA activity, inhibited pseudopod formation and chemotactic cell migration. These data demonstrate that spatial regulation of PKA via anchoring is an important facet of normal chemotactic cell movement.

  13. Aurora A kinase regulates proper spindle positioning in C. elegans and in human cells.

    Science.gov (United States)

    Kotak, Sachin; Afshar, Katayon; Busso, Coralie; Gönczy, Pierre

    2016-08-01

    Accurate spindle positioning is essential for error-free cell division. The one-cell Caenorhabditis elegans embryo has proven instrumental for dissecting mechanisms governing spindle positioning. Despite important progress, how the cortical forces that act on astral microtubules to properly position the spindle are modulated is incompletely understood. Here, we report that the PP6 phosphatase PPH-6 and its associated subunit SAPS-1, which positively regulate pulling forces acting on spindle poles, associate with the Aurora A kinase AIR-1 in C. elegans embryos. We show that acute inactivation of AIR-1 during mitosis results in excess pulling forces on astral microtubules. Furthermore, we uncover that AIR-1 acts downstream of PPH-6-SAPS-1 in modulating spindle positioning, and that PPH-6-SAPS-1 negatively regulates AIR-1 localization at the cell cortex. Moreover, we show that Aurora A and the PP6 phosphatase subunit PPP6C are also necessary for spindle positioning in human cells. There, Aurora A is needed for the cortical localization of NuMA and dynein during mitosis. Overall, our work demonstrates that Aurora A kinases and PP6 phosphatases have an ancient function in modulating spindle positioning, thus contributing to faithful cell division. PMID:27335426

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

    Science.gov (United States)

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

    2005-07-20

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

  15. Negative regulation of receptor tyrosine kinases: unexpected links to c-Cbl and receptor ubiquitylation

    Institute of Scientific and Technical Information of China (English)

    Chanan RUBIN; Gal GUR; Yosef YARDEN

    2005-01-01

    Intracellular signals mediated by the family of receptor tyrosine kinases play pivotal roles in morphogenesis, cell fate determination and pathogenesis. Precise control of signal amplitude and duration is critical for the fidelity and robustness of these processes. Activation of receptor tyrosine kinases by their cognate growth factors not only leads to propagation of the signal through various biochemical cascades, but also sets in motion multiple attenuation mechanisms that ultimately terminate the active state. Early attenuators pre-exist prior to receptor activation and they act to limit signal propagation. Subsequently, late attenuators, such as Lrig and Sprouty, are transcriptionally induced and further act to dampen the signal. Central to the process of signaling attenuation is the role of the E3 ubiquitin ligase c-Cbl. While Cblmediated processes of receptor ubiquitylation and endocytosis are relatively well understood, the links of Cbl to other negative regulators are just now beginning to be appreciated. Here we review some emerging interfaces between Cbl and the transcriptionally induced negative regulators Lrig and Sprouty.

  16. New Kinase Regulation Mechanism Found in HipBA: a Bacterial Persistence Switch

    Energy Technology Data Exchange (ETDEWEB)

    Evdokimov, A.; Voznesensky, I; Fennell, K; Anderson, M; Smith, J; Fisher, D

    2009-01-01

    Bacterial persistence is the ability of individual cells to randomly enter a period of dormancy during which the cells are protected against antibiotics. In Escherichia coli, persistence is regulated by the activity of a protein kinase HipA and its DNA-binding partner HipB, which is a strong inhibitor of both HipA activity and hip operon transcription. The crystal structure of the HipBA complex was solved by application of the SAD technique to a mercury derivative. In this article, the fortuitous and interesting effect of mercury soaks on the native HipBA crystals is discussed as well as the intriguing tryptophan-binding pocket found on the HipA surface. A HipA-regulation model is also proposed that is consistent with the available structural and biochemical data.

  17. New kinase regulation mechanism found in HipBA: a bacterial persistence switch.

    Science.gov (United States)

    Evdokimov, Artem; Voznesensky, Igor; Fennell, Kimberly; Anderson, Marie; Smith, James F; Fisher, Douglas A

    2009-08-01

    Bacterial persistence is the ability of individual cells to randomly enter a period of dormancy during which the cells are protected against antibiotics. In Escherichia coli, persistence is regulated by the activity of a protein kinase HipA and its DNA-binding partner HipB, which is a strong inhibitor of both HipA activity and hip operon transcription. The crystal structure of the HipBA complex was solved by application of the SAD technique to a mercury derivative. In this article, the fortuitous and interesting effect of mercury soaks on the native HipBA crystals is discussed as well as the intriguing tryptophan-binding pocket found on the HipA surface. A HipA-regulation model is also proposed that is consistent with the available structural and biochemical data. PMID:19622872

  18. Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases.

    Science.gov (United States)

    Sugden, Mary C; Holness, Mark J

    2006-07-01

    The mechanisms that control mammalian pyruvate dehydrogenase complex (PDC) activity include its phosphorylation (inactivation) by a family of pyruvate dehydrogenase kinases (PDKs 1 - 4). Here we review new developments in the regulation of the activities and expression of the PDKs, in particular PDK2 and PDK4, in relation to glucose and lipid homeostasis. This review describes recent advances relating to the acute and long-term modes of regulation of the PDKs, with particular emphasis on the regulatory roles of nuclear receptors including peroxisome proliferator-activated receptor (PPAR) alpha and Liver X receptor (LXR), PPAR gamma coactivator alpha (PGC-1alpha) and insulin, and the impact of changes in PDK activity and expression in glucose and lipid homeostasis. Since PDK4 may assist in lipid clearance when there is an imbalance between lipid delivery and oxidation, it may represent an attractive target for interventions aimed at rectifying abnormal lipid as well as glucose homeostasis in disease states. PMID:17132539

  19. Leptin stimulates pituitary prolactin release through an extracellular signal-regulated kinase-dependent pathway

    DEFF Research Database (Denmark)

    Tipsmark, Christian K; Strom, Christina N; Bailey, Sean T;

    2008-01-01

    of the actions of leptin are mediated by the activation of extracellular signal-regulated kinase (ERK1/2) but nothing is known about the cellular mechanisms by which leptin might regulate PRL secretion in vertebrates. We therefore tested whether ERK1/2 might be involved in the leptin PRL response and found...... that the ERK inhibitor, PD98059, hindered leptin-induced PRL release. We further analyzed leptin response by quantifying tyrosine and threonine phosphorylation of ERK1/2 using western blots. One hour incubation with leptin induced a concentration-dependent increase in phosphorylated, and thus active, ERK1/2....... Our data show that leptin is a powerful stimulator of in vitro PRL release and that its actions occur in part through stimulation of ERK1/2....

  20. Diacylglycerol Kinases: Regulated Controllers of T Cell Activation, Function, and Development

    Directory of Open Access Journals (Sweden)

    Gary A. Koretzky

    2013-03-01

    Full Text Available Diacylglycerol kinases (DGKs are a diverse family of enzymes that catalyze the conversion of diacylglycerol (DAG, a crucial second messenger of receptor-mediated signaling, to phosphatidic acid (PA. Both DAG and PA are bioactive molecules that regulate a wide set of intracellular signaling proteins involved in innate and adaptive immunity. Clear evidence points to a critical role for DGKs in modulating T cell activation, function, and development. More recently, studies have elucidated factors that control DGK function, suggesting an added complexity to how DGKs act during signaling. This review summarizes the available knowledge of the function and regulation of DGK isoforms in signal transduction with a particular focus on T lymphocytes.

  1. Brain-derived neurotrophic factor activation of extracellular signal-regulated kinase is autonomous from the dominant extrasynaptic NMDA receptor extracellular signal-regulated kinase shutoff pathway.

    Science.gov (United States)

    Mulholland, P J; Luong, N T; Woodward, J J; Chandler, L J

    2008-01-24

    NMDA receptors bidirectionally modulate extracellular signal-regulated kinase (ERK) through the coupling of synaptic NMDA receptors to an ERK activation pathway that is opposed by a dominant ERK shutoff pathway thought to be coupled to extrasynaptic NMDA receptors. In the present study, synaptic NMDA receptor activation of ERK in rat cortical cultures was partially inhibited by the highly selective NR2B antagonist Ro25-6981 (Ro) and the less selective NR2A antagonist NVP-AAM077 (NVP). When Ro and NVP were added together, inhibition appeared additive and equal to that observed with the NMDA open-channel blocker MK-801. Consistent with a selective coupling of extrasynaptic NMDA receptors to the dominant ERK shutoff pathway, pre-block of synaptic NMDA receptors with MK-801 did not alter the inhibitory effect of bath-applied NMDA on ERK activity. Lastly, in contrast to a complete block of synaptic NMDA receptor activation of ERK by extrasynaptic NMDA receptors, activation of extrasynaptic NMDA receptors had no effect upon ERK activation by brain-derived neurotrophic factor. These results suggest that the synaptic NMDA receptor ERK activation pathway is coupled to both NR2A and NR2B containing receptors, and that the extrasynaptic NMDA receptor ERK inhibitory pathway is not a non-selective global ERK shutoff.

  2. The structure of arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress

    KAUST Repository

    Yunta, Cristina

    2011-11-01

    SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases. © 2011 Elsevier Ltd. All rights reserved.

  3. Wheat CBL-interacting protein kinase 25 negatively regulates salt tolerance in transgenic wheat.

    Science.gov (United States)

    Jin, Xia; Sun, Tao; Wang, Xiatian; Su, Peipei; Ma, Jingfei; He, Guangyuan; Yang, Guangxiao

    2016-01-01

    CBL-interacting protein kinases are involved in plant responses to abiotic stresses, including salt stress. However, the negative regulating mechanism of this gene family in response to salinity is less reported. In this study, we evaluated the role of TaCIPK25 in regulating salt response in wheat. Under conditions of high salinity, TaCIPK25 expression was markedly down-regulated in roots. Overexpression of TaCIPK25 resulted in hypersensitivity to Na(+) and superfluous accumulation of Na(+) in transgenic wheat lines. TaCIPK25 expression did not decline in transgenic wheat and remained at an even higher level than that in wild-type wheat controls under high-salinity treatment. Furthermore, transmembrane Na(+)/H(+) exchange was impaired in the root cells of transgenic wheat. These results suggested that TaCIPK25 negatively regulated salt response in wheat. Additionally, yeast-one-hybrid, β-glucuronidase activity and DNA-protein-interaction-enzyme-linked-immunosorbent assays showed that the transcription factor TaWRKY9 bound W-box in the TaCIPK25 promoter region. Quantitative real-time polymerase chain reaction assays showed concomitantly inverted expression patterns of TaCIPK25 and TaWRKY9 in wheat roots under salt treatment, ABA application and inhibition of endogenous ABA condition. Overall, based on our results, in a salt stress condition, the negative salt response in wheat involved TaCIPK25 with the expression regulated by TaWRKY9. PMID:27358166

  4. Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2)

    DEFF Research Database (Denmark)

    Theilade, Juliane; Lerche Hansen, Jakob; Haunsø, Stig;

    2002-01-01

    G protein-coupled receptor kinase 2 (GRK2) phosphorylates G protein-coupled receptors resulting in uncoupling from G proteins. Receptors modulate GRK2 expression, however the mechanistic basis for this effect is largely unknown. Here we report a novel mechanism by which receptors use...

  5. Molecular Physiology of SPAK and OSR1: Two Ste20-Related Protein Kinases Regulating Ion Transport

    OpenAIRE

    Gagnon, Kenneth B; Delpire, Eric

    2012-01-01

    SPAK (Ste20-related proline alanine rich kinase) and OSR1 (oxidative stress responsive kinase) are members of the germinal center kinase VI sub-family of the mammalian Ste20 (Sterile20)-related protein kinase family. Although there are 30 enzymes in this protein kinase family, their conservation across the fungi, plant and animal kingdom confirms their evolutionary importance. Already, a large volume of work has accumulated on the tissue distribution, binding partners, signaling cascades, and...

  6. Complement receptor-3 negatively regulates the phagocytosis of degenerated myelin through tyrosine kinase Syk and cofilin

    Directory of Open Access Journals (Sweden)

    Hadas Smadar

    2012-07-01

    Full Text Available Abstract Background Intact myelin, which normally surrounds axons, breaks down in Wallerian degeneration following axonal injury and during neurodegenerative diseases such as multiple sclerosis. Clearance of degenerated myelin by phagocytosis is essential since myelin impedes repair and exacerbates damage. CR3 (complement receptor-3 is a principal phagocytic receptor in myelin phagocytosis. We studied how tyrosine kinase Syk (spleen tyrosine kinase and cofilin control phagocytosis of degenerated myelin by CR3 in microglia and macrophages. Syk is a non-receptor tyrosine kinase that CR3 recruits to convey cellular functions. Cofilin is an actin-depolymerizing protein that controls F-actin (filamentous actin remodeling (i.e., disassembly and reassembly by shifting between active unphosphorylated and inactive phosphorylated states. Results Syk was continuously activated during prolonged phagocytosis. Phagocytosis increased when Syk activity and expression were reduced, suggesting that normally Syk down regulates CR3-mediated myelin phagocytosis. Levels of inactive p-cofilin (phosphorylated cofilin decreased transiently during prolonged phagocytosis. In contrast, p-cofilin levels decreased continuously when Syk activity and expression were continuously reduced, suggesting that normally Syk advances the inactive state of cofilin. Observations also revealed inverse relationships between levels of phagocytosis and levels of inactive p-cofilin, suggesting that active unphosphorylated cofilin advances phagocytosis. Active cofilin could advance phagocytosis by promoting F-actin remodeling, which supports the production of membrane protrusions (e.g., filopodia, which, as we also revealed, are instrumental in myelin phagocytosis. Conclusions CR3 both activates and downregulates myelin phagocytosis at the same time. Activation was previously documented. We presently demonstrate that downregulation is mediated through Syk, which advances the inactive

  7. Phosphorylation of the tumour suppressor Fat is regulated via interaction with its ligand Dachsous, and the kinase, Discs Overgrown

    OpenAIRE

    Sopko, Richelle; Silva, Elizabeth; Clayton, Lesley; Gardano, Laura; Barrios-Rodiles, Miriam; Wrana, Jeff; Varelas, Xaralabos; Arbouzova, Natalia I.; Shaw, Sanjeev; Saburi, Sakura; Matakatsu, Hitoshi; Blair, Seth; McNeill, Helen

    2009-01-01

    The Drosophila tumour suppressor gene fat encodes a large cadherin that regulates growth and a form of tissue organization known as planar cell polarity (PCP). Fat regulates growth via the Hippo kinase pathway [1–4], which controls expression of genes promoting cell proliferation and inhibiting apoptosis (reviewed in [5–11]). The Hippo pathway is highly conserved and is implicated in the regulation of mammalian growth and cancer development [12–18]. Genetic studies suggest that Fat activity i...

  8. Quantitative phosphoproteomics reveals SLP-76 dependent regulation of PAG and Src family kinases in T cells.

    Directory of Open Access Journals (Sweden)

    Lulu Cao

    Full Text Available The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76 plays a critical scaffolding role in T cell receptor (TCR signaling. As an adaptor protein that contains multiple protein-binding domains, SLP-76 interacts with many signaling molecules and links proximal receptor stimulation to downstream effectors. The function of SLP-76 in TCR signaling has been widely studied using the Jurkat human leukaemic T cell line through protein disruption or site-directed mutagenesis. However, a wide-scale characterization of SLP-76-dependant phosphorylation events is still lacking. Quantitative profiling of over a hundred tyrosine phosphorylation sites revealed new modes of regulation of phosphorylation of PAG, PI3K, and WASP while reconfirming previously established regulation of Itk, PLCγ, and Erk phosphorylation by SLP-76. The absence of SLP-76 also perturbed the phosphorylation of Src family kinases (SFKs Lck and Fyn, and subsequently a large number of SFK-regulated signaling molecules. Altogether our data suggests unique modes of regulation of positive and negative feedback pathways in T cells by SLP-76, reconfirming its central role in the pathway.

  9. Exon-centric regulation of pyruvate kinase M alternative splicing via mutually exclusive exons

    Institute of Scientific and Technical Information of China (English)

    Zhenxun Wang; Deblina Chatterjee; Hyun Yong Jeon; Martin Akerman; Matthew G. Vander Heiden; Lewis C. Cantley; Adrian R. Krainer

    2012-01-01

    Alternative splicing of the pyruvate kinase M gene (PK-M) can generate the M2 isoform and promote aerobic glycolysis and tumor growth.However,the cancer-specific alternative splicing regulation of PK-M is not completely understood.Here,we demonstrate that PK-M is regulated by reciprocal affects on the mutually exclusive exons 9 and 10,such that exon 9 is repressed and exon 10 is activated in cancer cells.Strikingly,exonic,rather than intronic,cis-elements are key determinants ef PK-M splicing isoform ratios.Using a systematic sub-exonic duplication approach,we identify a potent exonlc splicing enhancer in exon 10,which differs from its homologous counterpart in exon 9 by only two nucleotides.We identify SRSF3 as one of the cognate factors,and show that this serine/arginine-rich protein activates exon 10 and mediates changes in glucose metabolism.These findings provide mechanistic insights into the complex regulation of alternative splicing of a key regulator of the Warburg effect,and also have implications for other genes with a similar pattern of alternative splicing.

  10. AAMP Regulates Endothelial Cell Migration and Angiogenesis Through RhoA/Rho Kinase Signaling.

    Science.gov (United States)

    Hu, Jianjun; Qiu, Juhui; Zheng, Yiming; Zhang, Tao; Yin, Tieying; Xie, Xiang; Wang, Guixue

    2016-05-01

    Angiogenesis is a complicated process including endothelial cell proliferation, migration and tube formation. AAMP plays a role in regulating cell migration of multiple cell types. The purpose of this study was to investigate whether AAMP regulates angiogenesis, and to clarify the role of AAMP in the VEGF-induced angiogenesis. We found that AAMP expressed in multiple cell types and mainly localized in cytoplasm and membrane in vascular endothelial cells. Using tube formation assay in vitro and aortic ring assay, siRNA-mediated knockdown and antibody blockade of AAMP impaired VEGF-induced endothelial cell tube formation and aortic ring angiogenic sprouting. Mechanistic studies showed that AAMP expression was significantly upregulated by VEGF in a concentration and time-dependent manner. Moreover, VEGF recruited AAMP to the cell membrane protrusions. AAMP regulates angiogenesis by mediating the spreading and migration of vascular endothelial cells. AAMP knock-down reduced VEGF-induced actin stress fibers and collagen gel contraction. Furthermore, we identified RhoA/Rho kinase signaling as an important factor that contributes to the action of AAMP in regulating endothelial cell migration and angiogenesis. Altogether, these data demonstrated the critical role of AAMP in angiogenesis and suggested blocking AAMP could serve as a potential therapeutic strategy for angiogenesis-related diseases. PMID:26350504

  11. Regulation of p73 by Hck through kinase-dependent and independent mechanisms

    Directory of Open Access Journals (Sweden)

    Radha Vegesna

    2007-05-01

    Full Text Available Abstract Background p73, a p53 family member is a transcription factor that plays a role in cell cycle, differentiation and apoptosis. p73 is regulated through post translational modifications and protein interactions. c-Abl is the only known tyrosine kinase that phosphorylates and activates p73. Here we have analyzed the role of Src family kinases, which are involved in diverse signaling pathways, in regulating p73. Results Exogenously expressed as well as cellular Hck and p73 interact in vivo. In vitro binding assays show that SH3 domain of Hck interacts with p73. Co-expression of p73 with Hck or c-Src in mammalian cells resulted in tyrosine phosphorylation of p73. Using site directed mutational analysis, we determined that Tyr-28 was the major site of phosphorylation by Hck and c-Src, unlike c-Abl which phosphorylates Tyr-99. In a kinase dependent manner, Hck co-expression resulted in stabilization of p73 protein in the cytoplasm. Activation of Hck in HL-60 cells resulted in tyrosine phosphorylation of endogenous p73. Both exogenous and endogenous Hck localize to the nuclear as well as cytoplasmic compartment, just as does p73. Ectopically expressed Hck repressed the transcriptional activity of p73 as determined by promoter assays and semi-quantitative RT-PCR analysis of the p73 target, Ipaf and MDM2. SH3 domain- dependent function of Hck was required for its effect on p73 activity, which was also reflected in its ability to inhibit p73-mediated apoptosis. We also show that Hck interacts with Yes associated protein (YAP, a transcriptional co-activator of p73, and shRNA mediated knockdown of YAP protein reduces p73 induced Ipaf promoter activation. Conclusion We have identified p73 as a novel substrate and interacting partner of Hck and show that it regulates p73 through mechanisms that are dependent on either catalytic activity or protein interaction domains. Hck-SH3 domain-mediated interactions play an important role in the inhibition of p73

  12. Janus kinases and Src family kinases in the regulation of EGF-induced vimentin expression in MDA-MB-468 breast cancer cells.

    Science.gov (United States)

    Stewart, Teneale A; Azimi, Iman; Brooks, Andrew J; Thompson, Erik W; Roberts-Thomson, Sarah J; Monteith, Gregory R

    2016-07-01

    Epithelial-mesenchymal transition (EMT) is an important process associated with the metastasis of breast cancer cells. Members of the Janus kinases (JAKs) and Src family kinases (SFKs) are implicated in the regulation of an invasive phenotype in various cancer cell types. Using the pharmacological inhibitors JAK Inhibitor I (a pan-JAK inhibitor) and PP2 we investigated the role of the JAKs and SFKs, respectively, in the regulation of EMT markers in the MDA-MB-468 breast cancer cell line model of epidermal growth factor (EGF)-induced EMT. We identified selective inhibition of EGF induction of the mesenchymal marker vimentin by PP2 and JAK Inhibitor I. The effect of JAK Inhibitor I on vimentin protein induction occurred at a concentration lower than that required to significantly inhibit EGF-mediated signal transducer and activator of transcription 3 (STAT3)-phosphorylation, suggesting involvement of a STAT3-independent mechanism of EGF-induced vimentin regulation by JAKs. Despite our identification of a role for the JAK family in EGF-induced vimentin protein expression, siRNA-mediated silencing of each member of the JAK family was unable to phenocopy pharmacological inhibition, indicating potential redundancy among the JAK family members in this pathway. While SFKs and JAKs do not represent global regulators of the EMT phenotype, our findings have identified a role for members of these signaling pathways in the regulation of EGF-induced vimentin expression in the MDA-MB-468 breast cancer cell line. PMID:27163529

  13. A forward genetic screen reveals that calcium-dependent protein kinase 3 regulates egress in Toxoplasma.

    Directory of Open Access Journals (Sweden)

    Erin Garrison

    Full Text Available Egress from the host cell is a crucial and highly regulated step in the biology of the obligate intracellular parasite, Toxoplasma gondii. Active egress depends on calcium fluxes and appears to be a crucial step in escaping the attack from the immune system and, potentially, in enabling the parasites to shuttle into appropriate cells for entry into the brain of the host. Previous genetic screens have yielded mutants defective in both ionophore-induced egress and ionophore-induced death. Using whole genome sequencing of one mutant and subsequent analysis of all mutants from these screens, we find that, remarkably, four independent mutants harbor a mis-sense mutation in the same gene, TgCDPK3, encoding a calcium-dependent protein kinase. All four mutations are predicted to alter key regions of TgCDPK3 and this is confirmed by biochemical studies of recombinant forms of each. By complementation we confirm a crucial role for TgCDPK3 in the rapid induction of parasite egress and we establish that TgCDPK3 is critical for formation of latent stages in the brains of mice. Genetic knockout of TgCDPK3 confirms a crucial role for this kinase in parasite egress and a non-essential role for it in the lytic cycle.

  14. Protein kinase D2 induces invasion of pancreatic cancer cells by regulating matrix metalloproteinases.

    Science.gov (United States)

    Wille, Christoph; Köhler, Conny; Armacki, Milena; Jamali, Arsia; Gössele, Ulrike; Pfizenmaier, Klaus; Seufferlein, Thomas; Eiseler, Tim

    2014-02-01

    Pancreatic cancer cell invasion, metastasis, and angiogenesis are major challenges for the development of novel therapeutic strategies. Protein kinase D (PKD) isoforms are involved in controlling tumor cell motility, angiogenesis, and metastasis. In particular PKD2 expression is up-regulated in pancreatic cancer, whereas PKD1 expression is lowered. We report that both kinases control pancreatic cancer cell invasive properties in an isoform-specific manner. PKD2 enhances invasion in three-dimensional extracellular matrix (3D-ECM) cultures by stimulating expression and secretion of matrix metalloproteinases 7 and 9 (MMP7/9), by which MMP7 is likely to act upstream of MMP9. Knockdown of MMP7/9 blocks PKD2-mediated invasion in 3D-ECM assays and in vivo using tumors growing on chorioallantois membranes. Furthermore, MMP9 enhances PKD2-mediated tumor angiogenesis by releasing extracellular matrix-bound vascular endothelial growth factor A, increasing its bioavailability and angiogenesis. Of interest, specific knockdown of PKD1 in PKD2-expressing pancreatic cancer cells further enhanced the invasive properties in 3D-ECM systems by generating a high-motility phenotype. Loss of PKD1 thus may be beneficial for tumor cells to enhance their matrix-invading abilities. In conclusion, we define for the first time PKD1 and 2 isoform-selective effects on pancreatic cancer cell invasion and angiogenesis, in vitro and in vivo, addressing PKD isoform specificity as a major factor for future therapeutic strategies. PMID:24336522

  15. Regulation of Connexin43 Function and Expression by Tyrosine Kinase 2.

    Science.gov (United States)

    Li, Hanjun; Spagnol, Gaelle; Zheng, Li; Stauch, Kelly L; Sorgen, Paul L

    2016-07-22

    Connexin43 (Cx43) assembly and degradation, the regulation of electrical and metabolic coupling, as well as modulating the interaction with other proteins, involve phosphorylation. Here, we identified and characterized the biological significance of a novel tyrosine kinase that phosphorylates Cx43, tyrosine kinase 2 (Tyk2). Activation of Tyk2 led to a decrease in Cx43 gap junction communication by increasing the turnover rate of Cx43 from the plasma membrane. Tyk2 directly phosphorylated Cx43 residues Tyr-247 and Tyr-265, leading to indirect phosphorylation on residues Ser-279/Ser-282 (MAPK) and Ser-368 (PKC). Although this phosphorylation pattern is similar to what has been observed following Src activation, the response caused by Tyk2 occurred when Src was inactive in NRK cells. Knockdown of Tyk2 at the permissive temperature (active v-Src) in LA-25 cells decreased Cx43 phosphorylation, indicating that although activation of Tyk2 and v-Src leads to phosphorylation of the same Cx43CT residues, they are not identical in level at each site. Additionally, angiotensin II activation of Tyk2 increased the intracellular protein level of Cx43 via STAT3. These findings indicate that, like Src, Tyk2 can also inhibit gap junction communication by phosphorylating Cx43. PMID:27235399

  16. A PI3-kinase-mediated negative feedback regulates neuronal excitability.

    Directory of Open Access Journals (Sweden)

    Eric Howlett

    2008-11-01

    Full Text Available Use-dependent downregulation of neuronal activity (negative feedback can act as a homeostatic mechanism to maintain neuronal activity at a particular specified value. Disruption of this negative feedback might lead to neurological pathologies, such as epilepsy, but the precise mechanisms by which this feedback can occur remain incompletely understood. At one glutamatergic synapse, the Drosophila neuromuscular junction, a mutation in the group II metabotropic glutamate receptor gene (DmGluRA increased motor neuron excitability by disrupting an autocrine, glutamate-mediated negative feedback. We show that DmGluRA mutations increase neuronal excitability by preventing PI3 kinase (PI3K activation and consequently hyperactivating the transcription factor Foxo. Furthermore, glutamate application increases levels of phospho-Akt, a product of PI3K signaling, within motor nerve terminals in a DmGluRA-dependent manner. Finally, we show that PI3K increases both axon diameter and synapse number via the Tor/S6 kinase pathway, but not Foxo. In humans, PI3K and group II mGluRs are implicated in epilepsy, neurofibromatosis, autism, schizophrenia, and other neurological disorders; however, neither the link between group II mGluRs and PI3K, nor the role of PI3K-dependent regulation of Foxo in the control of neuronal excitability, had been previously reported. Our work suggests that some of the deficits in these neurological disorders might result from disruption of glutamate-mediated homeostasis of neuronal excitability.

  17. Peripheral motor neuropathy is associated with defective kinase regulation of the KCC3 cotransporter.

    Science.gov (United States)

    Kahle, Kristopher T; Flores, Bianca; Bharucha-Goebel, Diana; Zhang, Jinwei; Donkervoort, Sandra; Hegde, Madhuri; Hussain, Gulnaz; Duran, Daniel; Liang, Bo; Sun, Dandan; Bönnemann, Carsten G; Delpire, Eric

    2016-01-01

    Using exome sequencing, we identified a de novo mutation (c.2971A>G; T991A) in SLC12A6, the gene encoding the K(+)-Cl(-) cotransporter KCC3, in a patient with an early-onset, progressive, and severe peripheral neuropathy primarily affecting motor neurons. Normally, the WNK kinase-dependent phosphorylation of T(991) tonically inhibits KCC3; however, cell swelling triggers Thr(991) dephosphorylation to activate the transporter and restore cell volume. KCC3 T991A mutation in patient cells abolished Thr(991) phosphorylation, resulted in constitutive KCC3 activity, and compromised cell volume homeostasis. KCC3(T991A/T991A) mutant mice exhibited constitutive KCC3 activity and recapitulated aspects of the clinical, electrophysiological, and histopathological findings of the patient. These results suggest that the function of the peripheral nervous system depends on finely tuned, kinase-regulated KCC3 activity and implicate abnormal cell volume homeostasis as a previously unreported mechanism of axonal degeneration. PMID:27485015

  18. Inositol 1,4,5-trisphosphate 3-kinases: functions and regulations

    Institute of Scientific and Technical Information of China (English)

    Hui Jun XIA; Guang YANG

    2005-01-01

    Inositol 1,4,5-trisphosphate 3-kinase (IP3 3-kinase/IP3K) plays an important role in signal transduction in animal cells by phosphorylating inositol 1,4,5-trisphosphate (IP3) to inositol 1,3,4,5-tetrakisphosphate (IP4). Both IP3 and IP4 are critical second messengers which regulate calcium (Ca2+) homeostasis. Mammalian IP3Ks are involved in many biological processes, including brain development, memory, learning and so on. It is widely reported that Ca2+ is a canonical second messenger in higher plants. Therefore, plant IP3K should also play a crucial role in plant development. Recently,we reported the identification of plant IP3K gene (AtIpk2β/AtIP3K) from Arabidopsis thaliana and its characterization.Here, we summarize the molecular cloning, biochemical properties and biological functions of IP3Ks from animal, yeast and plant. This review also discusses potential functions of IP3Ks in signaling crosstalk, inositol phosphate metabolism,gene transcriptional control and so on.

  19. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Pan Shiow-Lin

    2009-05-01

    Full Text Available Abstract In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1 in denbinobin-induced apoptosis in human lung adenocarcinoma (A549 cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN, two antioxidants (N-acetyl-L-cysteine (NAC and glutathione (GSH, a c-Jun N-terminal kinase (JNK inhibitor (SP600125, and an activator protein-1 (AP-1 inhibitor (curcumin. Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

  20. Nemo-like kinase is a novel regulator of spinal and bulbar muscular atrophy.

    Science.gov (United States)

    Todd, Tiffany W; Kokubu, Hiroshi; Miranda, Helen C; Cortes, Constanza J; La Spada, Albert R; Lim, Janghoo

    2015-01-01

    Spinal and bulbar muscular atrophy (SBMA) is a progressive neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR) protein. Despite extensive research, the exact pathogenic mechanisms underlying SBMA remain elusive. In this study, we present evidence that Nemo-like kinase (NLK) promotes disease pathogenesis across multiple SBMA model systems. Most remarkably, loss of one copy of Nlk rescues SBMA phenotypes in mice, including extending lifespan. We also investigated the molecular mechanisms by which NLK exerts its effects in SBMA. Specifically, we have found that NLK can phosphorylate the mutant polyglutamine-expanded AR, enhance its aggregation, and promote AR-dependent gene transcription by regulating AR-cofactor interactions. Furthermore, NLK modulates the toxicity of a mutant AR fragment via a mechanism that is independent of AR-mediated gene transcription. Our findings uncover a crucial role for NLK in controlling SBMA toxicity and reveal a novel avenue for therapy development in SBMA. PMID:26308581

  1. The TAM family receptor tyrosine kinase TYRO3 is a negative regulator of type 2 immunity

    Science.gov (United States)

    Chan, Pamela Y.; Carrera Silva, Eugenio A.; De Kouchkovsky, Dimitri; Joannas, Leonel D.; Hao, Liming; Hu, Donglei; Huntsman, Scott; Eng, Celeste; Licona-Limón, Paula; Weinstein, Jason S.; Herbert, De’Broski R.; Craft, Joseph E.; Flavell, Richard A.; Repetto, Silvia; Correale, Jorge; Burchard, Esteban G.; Torgerson, Dara G.; Ghosh, Sourav; Rothlin, Carla V.

    2016-01-01

    Host responses against metazoan parasites or an array of environmental substances elicit type 2 immunity. Despite its protective function, type 2 immunity also drives allergic diseases. The mechanisms that regulate the magnitude of the type 2 response remain largely unknown. Here, we show that genetic ablation of a receptor tyrosine kinase encoded by Tyro3 in mice or the functional neutralization of its ortholog in human dendritic cells resulted in enhanced type 2 immunity. Furthermore, the TYRO3 agonist PROS1 was induced in T cells by the quintessential type 2 cytokine, interleukin-4. T cell–specific Pros1 knockouts phenocopied the loss of Tyro3. Thus, a PROS1-mediated feedback from adaptive immunity engages a rheostat, TYRO3, on innate immune cells to limit the intensity of type 2 responses. PMID:27034374

  2. Ras-mutant cancer cells display B-Raf binding to Ras that activates extracellular signal-regulated kinase and is inhibited by protein kinase A phosphorylation.

    Science.gov (United States)

    Li, Yanping; Takahashi, Maho; Stork, Philip J S

    2013-09-20

    The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.

  3. Integrin-linked kinase (ILK) modulates wound healing through regulation of hepatocyte growth factor (HGF)

    International Nuclear Information System (INIS)

    Integrin-linked kinase (ILK) is an intracellular effector of cell–matrix interactions and regulates many cellular processes, including growth, proliferation, survival, differentiation, migration, invasion and angiogenesis. The present work analyzes the role of ILK in wound healing in adult animals using a conditional knock-out of the ILK gene generated with the tamoxifen-inducible Cre-lox system (CRE-LOX mice). Results show that ILK deficiency leads to retarded wound closure in skin. Intracellular mechanisms involved in this process were analyzed in cultured mouse embryonic fibroblast (MEF) isolated from CRE-LOX mice and revealed that wounding promotes rapid activation of phosphatidylinositol 3-kinase (PI3K) and ILK. Knockdown of ILK resulted in a retarded wound closure due to a decrease in cellular proliferation and loss of HGF protein expression during the healing process, in vitro and in vivo. Alterations in cell proliferation and wound closure in ILK-deficient MEF or mice could be rescued by exogenous administration of human HGF. These data demonstrate, for the first time, that the activation of PI3K and ILK after skin wounding are critical for HGF-dependent tissue repair and wound healing. -- Highlights: ► ILK deletion results in decreased HGF expression and delayed scratch wound repair. ► PI3K/ILK/AKT pathway signals through HGF to regulate wound healing. ► An ILK-dependent increase in HGF expression is responsible for wound healing in vivo. ► ILK-KO mice are used to confirm the requirement for ILK function in wound healing. ► Human HGF treatment restores delayed wound closure in vitro and in vivo.

  4. Protein kinase D2 is an essential regulator of murine myoblast differentiation.

    Directory of Open Access Journals (Sweden)

    Alexander Kleger

    Full Text Available Muscle differentiation is a highly conserved process that occurs through the activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to generate new myofibers. A defined pattern of myogenic transcription factors is orchestrated during this process and is regulated via distinct signaling cascades involving various intracellular signaling pathways, including members of the protein kinase C (PKC family. The protein kinase D (PKD isoenzymes PKD1, -2, and -3, are prominent downstream targets of PKCs and phospholipase D in various biological systems including mouse and could hence play a role in muscle differentiation. In the present study, we used a mouse myoblast cell line (C2C12 as an in vitro model to investigate the role of PKDs, in particular PKD2, in muscle stem cell differentiation. We show that C2C12 cells express all PKD isoforms with PKD2 being highly expressed. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated during the initiation of mouse myoblast differentiation. Selective inhibition of PKCs or PKDs by pharmacological inhibitors blocked myotube formation. Depletion of PKD2 by shRNAs resulted in a marked inhibition of myoblast cell fusion. PKD2-depleted cells exhibit impaired regulation of muscle development-associated genes while the proliferative capacity remains unaltered. Vice versa forced expression of PKD2 increases myoblast differentiation. These findings were confirmed in primary mouse satellite cells where myotube fusion was also decreased upon inhibition of PKDs. Active PKD2 induced transcriptional activation of myocyte enhancer factor 2D and repression of Pax3 transcriptional activity. In conclusion, we identify PKDs, in particular PKD2, as a major mediator of muscle cell differentiation in vitro and thereby as a potential novel target for the modulation of muscle regeneration.

  5. The yeast Sks1p kinase signaling network regulates pseudohyphal growth and glucose response.

    Directory of Open Access Journals (Sweden)

    Cole Johnson

    2014-03-01

    Full Text Available The yeast Saccharomyces cerevisiae undergoes a dramatic growth transition from its unicellular form to a filamentous state, marked by the formation of pseudohyphal filaments of elongated and connected cells. Yeast pseudohyphal growth is regulated by signaling pathways responsive to reductions in the availability of nitrogen and glucose, but the molecular link between pseudohyphal filamentation and glucose signaling is not fully understood. Here, we identify the glucose-responsive Sks1p kinase as a signaling protein required for pseudohyphal growth induced by nitrogen limitation and coupled nitrogen/glucose limitation. To identify the Sks1p signaling network, we applied mass spectrometry-based quantitative phosphoproteomics, profiling over 900 phosphosites for phosphorylation changes dependent upon Sks1p kinase activity. From this analysis, we report a set of novel phosphorylation sites and highlight Sks1p-dependent phosphorylation in Bud6p, Itr1p, Lrg1p, Npr3p, and Pda1p. In particular, we analyzed the Y309 and S313 phosphosites in the pyruvate dehydrogenase subunit Pda1p; these residues are required for pseudohyphal growth, and Y309A mutants exhibit phenotypes indicative of impaired aerobic respiration and decreased mitochondrial number. Epistasis studies place SKS1 downstream of the G-protein coupled receptor GPR1 and the G-protein RAS2 but upstream of or at the level of cAMP-dependent PKA. The pseudohyphal growth and glucose signaling transcription factors Flo8p, Mss11p, and Rgt1p are required to achieve wild-type SKS1 transcript levels. SKS1 is conserved, and deletion of the SKS1 ortholog SHA3 in the pathogenic fungus Candida albicans results in abnormal colony morphology. Collectively, these results identify Sks1p as an important regulator of filamentation and glucose signaling, with additional relevance towards understanding stress-responsive signaling in C. albicans.

  6. Integrin-linked kinase (ILK) modulates wound healing through regulation of hepatocyte growth factor (HGF)

    Energy Technology Data Exchange (ETDEWEB)

    Serrano, Isabel; Diez-Marques, Maria L.; Rodriguez-Puyol, Manuel [Department of Physiology, University of Alcala, Alcala de Henares, Madrid (Spain); Red de Investigacion Renal Cooperativa (RedinRen) (Spain); Instituto Reina Sofia de Investigacion Nefrologica (Spain); Herrero-Fresneda, Inmaculada [Nephrology Unit, IDIBELL, Hospital de Bellvitge, Barcelona (Spain); Red de Investigacion Renal Cooperativa (RedinRen) (Spain); Garcia del Moral, Raimundo [Department of Pathology, University of Granada (Spain); Red de Investigacion Renal Cooperativa (RedinRen) (Spain); Dedhar, Shoukat [Department of Integrative Oncology, BC Cancer Research Center, Vancouver, BC (Canada); Ruiz-Torres, Maria P., E-mail: mpiedad.ruiz@uah.es [Department of Physiology, University of Alcala, Alcala de Henares, Madrid (Spain); Red de Investigacion Renal Cooperativa (RedinRen) (Spain); Instituto Reina Sofia de Investigacion Nefrologica (Spain); Rodriguez-Puyol, Diego [Nephrology Unit, Hospital Universitario Principe de Asturias, Alcala de Henares, Madrid (Spain); Red de Investigacion Renal Cooperativa (RedinRen) (Spain); Instituto Reina Sofia de Investigacion Nefrologica (Spain)

    2012-11-15

    Integrin-linked kinase (ILK) is an intracellular effector of cell-matrix interactions and regulates many cellular processes, including growth, proliferation, survival, differentiation, migration, invasion and angiogenesis. The present work analyzes the role of ILK in wound healing in adult animals using a conditional knock-out of the ILK gene generated with the tamoxifen-inducible Cre-lox system (CRE-LOX mice). Results show that ILK deficiency leads to retarded wound closure in skin. Intracellular mechanisms involved in this process were analyzed in cultured mouse embryonic fibroblast (MEF) isolated from CRE-LOX mice and revealed that wounding promotes rapid activation of phosphatidylinositol 3-kinase (PI3K) and ILK. Knockdown of ILK resulted in a retarded wound closure due to a decrease in cellular proliferation and loss of HGF protein expression during the healing process, in vitro and in vivo. Alterations in cell proliferation and wound closure in ILK-deficient MEF or mice could be rescued by exogenous administration of human HGF. These data demonstrate, for the first time, that the activation of PI3K and ILK after skin wounding are critical for HGF-dependent tissue repair and wound healing. -- Highlights: Black-Right-Pointing-Pointer ILK deletion results in decreased HGF expression and delayed scratch wound repair. Black-Right-Pointing-Pointer PI3K/ILK/AKT pathway signals through HGF to regulate wound healing. Black-Right-Pointing-Pointer An ILK-dependent increase in HGF expression is responsible for wound healing in vivo. Black-Right-Pointing-Pointer ILK-KO mice are used to confirm the requirement for ILK function in wound healing. Black-Right-Pointing-Pointer Human HGF treatment restores delayed wound closure in vitro and in vivo.

  7. Mps1 kinase regulates tumor cell viability via its novel role in mitochondria.

    Science.gov (United States)

    Zhang, X; Ling, Y; Guo, Y; Bai, Y; Shi, X; Gong, F; Tan, P; Zhang, Y; Wei, C; He, X; Ramirez, A; Liu, X; Cao, C; Zhong, H; Xu, Q; Ma, R Z

    2016-01-01

    Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells. PMID:27383047

  8. 5´AMP activated protein kinase α2 controls substrate metabolism during post-exercise recovery via regulation of pyruvate dehydrogenase kinase 4

    DEFF Research Database (Denmark)

    Fritzen, Andreas Mæchel; Lundsgaard, Anne-Marie; Jeppesen, Jacob;

    2015-01-01

    after prolonged exercise and during the following six hours post exercise in 5´AMP activated protein kinase (AMPK)α2 and α1 knock-out (KO) and wild type (WT) mice with free access to food. Substrate oxidation was similar during exercise at the same relative intensity between genotypes. During post...... in muscle pyruvate dehydrogenase kinase 4 (PDK4) mRNA expression in WT and AMPKα2 KO was observed following exercise, which is consistent with AMPKα2 -deficiency not affecting the exercise-induced activation of the PDK4 transcriptional regulators, HDAC4 and SIRT1. Interestingly, PDK4 protein content...... increased (63 %, P protein content, lower (P

  9. MUC1-C oncoprotein regulates glycolysis and pyruvate kinase M2 activity in cancer cells.

    Directory of Open Access Journals (Sweden)

    Michio Kosugi

    Full Text Available Aerobic glycolysis in cancer cells is regulated by multiple effectors that include Akt and pyruvate kinase M2 (PKM2. Mucin 1 (MUC1 is a heterodimeric glycoprotein that is aberrantly overexpressed by human breast and other carcinomas. Here we show that transformation of rat fibroblasts by the oncogenic MUC1-C subunit is associated with Akt-mediated increases in glucose uptake and lactate production, consistent with the stimulation of glycolysis. The results also demonstrate that the MUC1-C cytoplasmic domain binds directly to PKM2 at the B- and C-domains. Interaction between the MUC1-C cytoplasmic domain Cys-3 and the PKM2 C-domain Cys-474 was found to stimulate PKM2 activity. Conversely, epidermal growth factor receptor (EGFR-mediated phosphorylation of the MUC1-C cytoplasmic domain on Tyr-46 conferred binding to PKM2 Lys-433 and inhibited PKM2 activity. In human breast cancer cells, silencing MUC1-C was associated with decreases in glucose uptake and lactate production, confirming involvement of MUC1-C in the regulation of glycolysis. In addition, EGFR-mediated phosphorylation of MUC1-C in breast cancer cells was associated with decreases in PKM2 activity. These findings indicate that the MUC1-C subunit regulates glycolysis and that this response is conferred in part by PKM2. Thus, the overexpression of MUC1-C oncoprotein in diverse human carcinomas could be of importance to the Warburg effect of aerobic glycolysis.

  10. YihE Kinase Is a Central Regulator of Programmed Cell Death in Bacteria

    Directory of Open Access Journals (Sweden)

    Angella Dorsey-Oresto

    2013-02-01

    Full Text Available Stress-mediated programmed cell death (PCD in bacteria has recently attracted attention, largely because it raises novel possibilities for controlling pathogens. How PCD in bacteria is regulated to avoid population extinction due to transient, moderate stress remains a central question. Here, we report that the YihE protein kinase is a key regulator that protects Escherichia coli from antimicrobial and environmental stressors by antagonizing the MazEF toxin-antitoxin module. YihE was linked to a reactive oxygen species (ROS cascade, and a deficiency of yihE stimulated stress-induced PCD even after stress dissipated. YihE was partially regulated by the Cpx envelope stress-response system, which, along with MazF toxin and superoxide, has both protective and destructive roles that help bacteria make a live-or-die decision in response to stress. YihE probably acts early in the stress response to limit self-sustaining ROS production and PCD. Inhibition of YihE may provide a way of enhancing antimicrobial lethality and attenuating virulence.

  11. Protein Kinase C Regulates the Cell Surface Activity of Endothelin-Converting Enzyme-1.

    Science.gov (United States)

    Smith, A Ian; Lew, Rebecca A; Thomas, Walter G; Tochon-Danguy, Nathalie

    2006-09-01

    The potent vasoconstrictor endothelin is a 21 amino acid peptide whose principal physiological function is to regulate vascular tone. The generation of endothelin is crucially dependent on the local presence and activity of endothelin converting enzyme-1 (ECE-1) expressed on the surface of vascular endothelial cells. In this study, we have shown in endothelial cells that the enzyme is phosphorylated, and that phosphorylation is increased by phorbol ester stimulation of protein kinase C (PKC). Furthermore, by monitoring specific ECE-1 activity on the surface of live cells, we also show that following PKC activation, enzyme activity is significantly increased at the cell surface, where it is positioned to catalyse the generation of active endothelin. We believe this novel finding is unprecedented for a peptide processing enzyme. Indeed, this new knowledge regarding the control of endothelin production by regulating ECE-1 activity at the cell surface opens up a new area of endothelin biology and will provide novel insights into the physiology and pathophysiology of endothelin and endothelin-associated diseases. In addition, the information generated in these studies may provide valuable new insights into potential extra- and intracellular targets for the pharmacological and perhaps even therapeutic regulation of endothelin production and thus vascular tone. PMID:19617920

  12. The kinase DYRK1A reciprocally regulates the differentiation of Th17 and regulatory T cells.

    Science.gov (United States)

    Khor, Bernard; Gagnon, John D; Goel, Gautam; Roche, Marly I; Conway, Kara L; Tran, Khoa; Aldrich, Leslie N; Sundberg, Thomas B; Paterson, Alison M; Mordecai, Scott; Dombkowski, David; Schirmer, Melanie; Tan, Pauline H; Bhan, Atul K; Roychoudhuri, Rahul; Restifo, Nicholas P; O'Shea, John J; Medoff, Benjamin D; Shamji, Alykhan F; Schreiber, Stuart L; Sharpe, Arlene H; Shaw, Stanley Y; Xavier, Ramnik J

    2015-01-01

    The balance between Th17 and T regulatory (Treg) cells critically modulates immune homeostasis, with an inadequate Treg response contributing to inflammatory disease. Using an unbiased chemical biology approach, we identified a novel role for the dual specificity tyrosine-phosphorylation-regulated kinase DYRK1A in regulating this balance. Inhibition of DYRK1A enhances Treg differentiation and impairs Th17 differentiation without affecting known pathways of Treg/Th17 differentiation. Thus, DYRK1A represents a novel mechanistic node at the branch point between commitment to either Treg or Th17 lineages. Importantly, both Treg cells generated using the DYRK1A inhibitor harmine and direct administration of harmine itself potently attenuate inflammation in multiple experimental models of systemic autoimmunity and mucosal inflammation. Our results identify DYRK1A as a physiologically relevant regulator of Treg cell differentiation and suggest a broader role for other DYRK family members in immune homeostasis. These results are discussed in the context of human diseases associated with dysregulated DYRK activity. PMID:25998054

  13. GSK-3 and CK2 Kinases Converge on Timeless to Regulate the Master Clock

    Directory of Open Access Journals (Sweden)

    Deniz Top

    2016-07-01

    Full Text Available The molecular clock relies on a delayed negative feedback loop of transcriptional regulation to generate oscillating gene expression. Although the principal components of the clock are present in all circadian neurons, different neuronal clusters have varying effects on rhythmic behavior, suggesting that the clocks they house are differently regulated. Combining biochemical and genetic techniques in Drosophila, we identify a phosphorylation program native to the master pacemaker neurons that regulates the timing of nuclear accumulation of the Period/Timeless repressor complex. GSK-3/SGG binds and phosphorylates Period-bound Timeless, triggering a CK2-mediated phosphorylation cascade. Mutations that block the hierarchical phosphorylation of Timeless in vitro also delay nuclear accumulation in both tissue culture and in vivo and predictably change rhythmic behavior. This two-kinase phosphorylation cascade is anatomically restricted to the eight master pacemaker neurons, distinguishing the regulatory mechanism of the molecular clock within these neurons from the other clocks that cooperate to govern behavioral rhythmicity.

  14. The kinase DYRK1A reciprocally regulates the differentiation of Th17 and regulatory T cells

    Science.gov (United States)

    Khor, Bernard; Gagnon, John D; Goel, Gautam; Roche, Marly I; Conway, Kara L; Tran, Khoa; Aldrich, Leslie N; Sundberg, Thomas B; Paterson, Alison M; Mordecai, Scott; Dombkowski, David; Schirmer, Melanie; Tan, Pauline H; Bhan, Atul K; Roychoudhuri, Rahul; Restifo, Nicholas P; O'Shea, John J; Medoff, Benjamin D; Shamji, Alykhan F; Schreiber, Stuart L; Sharpe, Arlene H; Shaw, Stanley Y; Xavier, Ramnik J

    2015-01-01

    The balance between Th17 and T regulatory (Treg) cells critically modulates immune homeostasis, with an inadequate Treg response contributing to inflammatory disease. Using an unbiased chemical biology approach, we identified a novel role for the dual specificity tyrosine-phosphorylation-regulated kinase DYRK1A in regulating this balance. Inhibition of DYRK1A enhances Treg differentiation and impairs Th17 differentiation without affecting known pathways of Treg/Th17 differentiation. Thus, DYRK1A represents a novel mechanistic node at the branch point between commitment to either Treg or Th17 lineages. Importantly, both Treg cells generated using the DYRK1A inhibitor harmine and direct administration of harmine itself potently attenuate inflammation in multiple experimental models of systemic autoimmunity and mucosal inflammation. Our results identify DYRK1A as a physiologically relevant regulator of Treg cell differentiation and suggest a broader role for other DYRK family members in immune homeostasis. These results are discussed in the context of human diseases associated with dysregulated DYRK activity. DOI: http://dx.doi.org/10.7554/eLife.05920.001 PMID:25998054

  15. Computational Modelling of the Metabolic States Regulated by the Kinase Akt

    Directory of Open Access Journals (Sweden)

    Ettore eMosca

    2012-11-01

    Full Text Available Signal transduction pathways and gene regulation determine a major reorganization of metabolic activities in order to support cell proliferation. Protein Kinase B (PKB, also known as Akt, participates in the PI3K/Akt/mTOR pathway, a master regulator of aerobic glycolysis and cellular biosynthesis, two activities shown by both normal and cancer proliferating cells. Not surprisingly considering its relevance for cellular metabolism, Akt/PKB is often found hyperactive in cancer cells. In the last decade, many efforts have been made to improve the understanding of the control of glucose metabolism and the identification of a therapeutic window between proliferating cancer cells and proliferating normal cells. In this context, we have modelled the link between the PI3K/Akt/mTOR pathway, glycolysis, lactic acid production and nucleotide biosynthesis. We used a computational model in order to compare two metabolic states generated by the specific variation of the metabolic fluxes regulated by the activity of the PI3K/Akt/mTOR pathway. One of the two states represented the metabolism of a growing cancer cell characterised by aerobic glycolysis and cellular biosynthesis, while the other state represented the same metabolic network with a reduced glycolytic rate and a higher mitochondrial pyruvate metabolism, as reported in literature in relation to the activity of the PI3K/Akt/mTOR. Some steps that link glycolysis and pentose phosphate pathway revealed their importance for controlling the dynamics of cancer glucose metabolism.

  16. The Endocytic Fate of the Transferrin Receptor Is Regulated by c-Abl Kinase.

    Science.gov (United States)

    Cao, Hong; Schroeder, Barbara; Chen, Jing; Schott, Micah B; McNiven, Mark A

    2016-08-01

    Clathrin-mediated endocytosis of transferrin (Tf) and its cognate receptor (TfR1) is a central pathway supporting the uptake of trophic iron. It has generally been assumed that this is a constitutive process. However, we have reported that the non-receptor tyrosine kinase, Src, is activated by Tf to facilitate the internalization of the Tf-TfR1 ligand-receptor complex. As an extension of these findings, we have tested whether subsequent trafficking steps might be regulated by additional kinase-dependent cascades, and we observed a significant endocytic block by inhibiting c-Abl kinase by a variety of methods. Importantly, Tf internalization was reduced significantly in all of these cell models and could be restored by re-expression of WT c-Abl. Surprisingly, this attenuated Tf-TfR1 endocytosis was due to a substantial drop in both the surface and total cellular receptor levels. Additional studies with the LDL receptor showed a similar effect. Surprisingly, immunofluorescence microscopy of imatinib-treated cells revealed a marked colocalization of internalized TfR1 with late endosomes/lysosomes, whereas attenuating the lysosome function with several inhibitors reduced this receptor loss. Importantly, inhibition of c-Abl resulted in a striking redistribution of the chaperone Hsc70 from a diffuse cytosolic localization to an association with the TfR1 at the late endosome-lysosome. Pharmacological inhibition of Hsc70 ATPase activity in cultured cells by the drug VER155008 prevents this chaperone-receptor interaction, resulting in an accumulation of the TfR1 in the early endosome. Thus, inhibition of c-Abl minimizes receptor recycling pathways and results in chaperone-dependent trafficking of the TfR1 to the lysosome for degradation. These findings implicate a novel role for c-Abl and Hsc70 as an unexpected regulator of Hsc70-mediated transport of trophic receptor cargo between the early and late endosomal compartments. PMID:27226592

  17. The Mitogen-Activated Protein Kinase Kinase VdPbs2 of Verticillium dahliae Regulates Microsclerotia Formation, Stress Response, and Plant Infection

    Science.gov (United States)

    Tian, Longyan; Wang, Yonglin; Yu, Jun; Xiong, Dianguang; Zhao, Hengjun; Tian, Chengming

    2016-01-01

    Verticillium dahliae, a ubiquitous phytopathogenic fungus, forms resting structures, known as microsclerotia that play crucial roles in Verticillium wilt diseases. VdHog1, a mitogen-activated protein kinase (MAPK), controls microsclerotia formation, virulence, and stress response in V. dahliae. In this study, we present detailed evidence that the conserved upstream component of VdHog1, VdPbs2, is a key regulator of microsclerotia formation, oxidative stress and fungicide response and plant virulence in V. dahliae. We identified VdPbs2, homologous to the yeast MAPK kinase Pbs2. Similar to the VdHog1 deletion mutant, VdPbs2 deletion strains exhibited delayed melanin synthesis and reduced formation of microsclerotia. When exposed to stresses, VdPbs2 mutants were more sensitive than the wild type to osmotic agents and peroxide, but more resistant to inhibitors of cell wall synthesis and some fungicides. Finally, VdPbs2 deletion mutants exhibited reduced virulence on smoke tree and tobacco seedlings. When taken together, we implicate that VdPbs2 and VdHog1 function in a cascade that regulates microsclerotia formation and virulence, but not all VdHog1 dependent functions are VdPbs2 regulated. This study thus provides novel insights into the signal transduction mechanisms that regulate microsclerotia formation and pathogenesis in this fungus. PMID:27729908

  18. Short-term regulation of NHE3 by EGF and protein kinase C but not protein kinase A involves vesicle trafficking in epithelial cells and fibroblasts.

    Science.gov (United States)

    Donowitz, M; Janecki, A; Akhter, S; Cavet, M E; Sanchez, F; Lamprecht, G; Zizak, M; Kwon, W L; Khurana, S; Yun, C H; Tse, C M

    2000-01-01

    NHE3 is an intestinal epithelial isoform Na+/H+ exchanger that is present in the brush border of small intestinal, colonic, and gallbladder Na(+)-absorbing epithelial cells. NHE3 is acutely up- and downregulated in response to some G protein-linked receptors, tyrosine kinase receptors, and protein kinases when studied in intact ileum, when stably expressed in PS120 fibroblasts, and in the few studies reported in the human colon cancer cell line Caco-2. In most cases this is due to changes in Vmax of NHE3, although in response to cAMP and squalamine there are also changes in the K'(H+)i of the exchanger. The mechanism of the Vmax regulation as shown by cell surface biotinylation and confocal microscopy in Caco-2 cells and biotinylation in PS120 cells involves changes in the amount of NHE3 on the plasma membrane. In addition, in some cases there are also changes in turnover number of the exchanger. In some cases, the change in amount of NHE3 in the plasma membrane is associated with a change in the amount of plasma membrane. A combination of biochemical studies and transport/inhibitor studies in intact ileum and Caco-2 cells demonstrated that the increase in brush border Na+/H+ exchange caused by acute exposure to EGF was mediated by PI 3-kinase. PI 3-kinase was also involved in FGF stimulation of NHE3 expressed in fibroblasts. Thus, NHE3 is another example of a transport protein that is acutely regulated in part by changing the amount of the transporter on the plasma membrane by a process that appears to involve vesicle trafficking and also to involve changes in turnover number. PMID:11193592

  19. Regulation of Ribosomal S6 Protein Kinase-p90rsk, Glycogen Synthase Kinase 3, and β-Catenin in Early Xenopus Development

    Science.gov (United States)

    Torres, Monica A.; Eldar-Finkelman, Hagit; Krebs, Edwin G.; Moon, Randall T.

    1999-01-01

    β-Catenin is a multifunctional protein that binds cadherins at the plasma membrane, HMG box transcription factors in the nucleus, and several cytoplasmic proteins that are involved in regulating its stability. In developing embryos and in some human cancers, the accumulation of β-catenin in the cytoplasm and subsequently the nuclei of cells may be regulated by the Wnt-1 signaling cascade and by glycogen synthase kinase 3 (GSK-3). This has increased interest in regulators of both GSK-3 and β-catenin. Searching for kinase activities able to phosphorylate the conserved, inhibitory-regulatory GSK-3 residue serine 9, we found p90rsk to be a potential upstream regulator of GSK-3. Overexpression of p90rsk in Xenopus embryos leads to increased steady-state levels of total β-catenin but not of the free soluble protein. Instead, p90rsk overexpression increases the levels of β-catenin in a cell fraction containing membrane-associated cadherins. Consistent with the lack of elevation of free β-catenin levels, ectopic p90rsk was unable to rescue dorsal cell fate in embryos ventralized by UV irradiation. We show that p90rsk is a downstream target of fibroblast growth factor (FGF) signaling during early Xenopus development, since ectopic FGF signaling activates both endogenous and overexpressed p90rsk. Moreover, overexpression of a dominant negative FGF receptor, which blocks endogenous FGF signaling, leads to decreased p90rsk kinase activity. Finally, we report that FGF inhibits endogenous GSK-3 activity in Xenopus embryos. We hypothesize that FGF and p90rsk play heretofore unsuspected roles in modulating GSK-3 and β-catenin. PMID:9891076

  20. Involvement of Phosphatidylinositol 3-kinase in the regulation of proline catabolism in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Anne-Sophie eLeprince

    2015-01-01

    Full Text Available Plant adaptation to abiotic stresses such as drought and salinity involves complex regulatory processes. Deciphering the signalling components that are involved in stress signal transduction and cellular responses is of importance to understand how plants cope with salt stress. Accumulation of osmolytes such as proline is considered to participate in the osmotic adjustment of plant cells to salinity. Proline accumulation results from a tight regulation between its biosynthesis and catabolism. Lipid signal components such as phospholipases C and D have previously been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. In this study, we demonstrate that proline metabolism is also regulated by class-III Phosphatidylinositol 3-kinase (PI3K, VPS34, which catalyses the formation of phosphatidylinositol 3-phosphate (PI3P from phosphatidylinositol. Using pharmacological and biochemical approaches, we show that the PI3K inhibitor, LY294002, affects PI3P levels in vivo and that it triggers a decrease in proline accumulation in response to salt treatment of A. thaliana seedlings. The lower proline accumulation is correlated with a lower transcript level of Pyrroline-5-carboxylate synthetase 1 biosynthetic enzyme and higher transcript and protein levels of Proline dehydrogenase 1 (ProDH1, a key-enzyme in proline catabolism. We also found that the ProDH1 expression is induced in a pi3k-hemizygous mutant, further demonstrating that PI3K is involved in the regulation of proline catabolism through transcriptional regulation of ProDH1. A broader metabolomic analysis indicates that LY294002 also reduced other metabolites, such as hydrophobic and aromatic amino acids and sugars like raffinose.

  1. AMP-Activated Protein Kinase Regulates Oxidative Metabolism in Caenorhabditis elegans through the NHR-49 and MDT-15 Transcriptional Regulators.

    Science.gov (United States)

    Moreno-Arriola, Elizabeth; El Hafidi, Mohammed; Ortega-Cuéllar, Daniel; Carvajal, Karla

    2016-01-01

    Cellular energy regulation relies on complex signaling pathways that respond to fuel availability and metabolic demands. Dysregulation of these networks is implicated in the development of human metabolic diseases such as obesity and metabolic syndrome. In Caenorhabditis elegans the AMP-activated protein kinase, AAK, has been associated with longevity and stress resistance; nevertheless its precise role in energy metabolism remains elusive. In the present study, we find an evolutionary conserved role of AAK in oxidative metabolism. Similar to mammals, AAK is activated by AICAR and metformin and leads to increased glycolytic and oxidative metabolic fluxes evidenced by an increase in lactate levels and mitochondrial oxygen consumption and a decrease in total fatty acids and lipid storage, whereas augmented glucose availability has the opposite effects. We found that these changes were largely dependent on the catalytic subunit AAK-2, since the aak-2 null strain lost the observed metabolic actions. Further results demonstrate that the effects due to AAK activation are associated to SBP-1 and NHR-49 transcriptional factors and MDT-15 transcriptional co-activator, suggesting a regulatory pathway that controls oxidative metabolism. Our findings establish C. elegans as a tractable model system to dissect the relationship between distinct molecules that play a critical role in the regulation of energy metabolism in human metabolic diseases.

  2. Up-regulation of bradykinin receptors in rat bronchi via I kappa B kinase-mediated inflammatory signaling pathway

    DEFF Research Database (Denmark)

    Lei, Ying; Zhang, Yaping; Cao, Yongxiao;

    2010-01-01

    IkappaB kinase (IKK)-mediated intracellular signaling mechanisms may be involved in airway hyperresponsiveness through up-regulation of bradykinin receptors. This study was designed to examine if organ culture of rat bronchial segments induces airway hyperresponsiveness to bradykinin and if inhib...

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

    Science.gov (United States)

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

    2003-01-01

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

  4. Structural basis for the regulation of maternal embryonic leucine zipper kinase.

    Directory of Open Access Journals (Sweden)

    Lu-Sha Cao

    Full Text Available MELK (maternal embryonic leucine zipper kinase, which is a member of the AMPK (AMP-activated protein kinase-related kinase family, plays important roles in diverse cellular processes and has become a promising drug target for certain cancers. However, the regulatory mechanism of MELK remains elusive. Here, we report the crystal structure of a fragment of human MELK that contains the kinase domain and ubiquitin-associated (UBA domain. The UBA domain tightly binds to the back of the kinase domain, which may contribute to the proper conformation and activity of the kinase domain. Interestingly, the activation segment in the kinase domain displays a unique conformation that contains an intramolecular disulfide bond. The structural and biochemical analyses unravel the molecular mechanisms for the autophosphorylation/activation of MELK and the dependence of its catalytic activity on reducing agents. Thus, our results may provide the basis for designing specific MELK inhibitors for cancer treatment.

  5. Proper Actin Ring Formation and Septum Constriction Requires Coordinated Regulation of SIN and MOR Pathways through the Germinal Centre Kinase MST-1

    Science.gov (United States)

    Heilig, Yvonne; Dettmann, Anne; Mouriño-Pérez, Rosa R.; Schmitt, Kerstin; Valerius, Oliver; Seiler, Stephan

    2014-01-01

    Nuclear DBF2p-related (NDR) kinases constitute a functionally conserved protein family of eukaryotic regulators that control cell division and polarity. In fungi, they function as effector kinases of the morphogenesis (MOR) and septation initiation (SIN) networks and are activated by pathway-specific germinal centre (GC) kinases. We characterized a third GC kinase, MST-1, that connects both kinase cascades. Genetic and biochemical interactions with SIN components and life cell imaging identify MST-1 as SIN-associated kinase that functions in parallel with the GC kinase SID-1 to activate the SIN-effector kinase DBF-2. SID-1 and MST-1 are both regulated by the upstream SIN kinase CDC-7, yet in an opposite manner. Aberrant cortical actomyosin rings are formed in Δmst-1, which resulted in mis-positioned septa and irregular spirals, indicating that MST-1-dependent regulation of the SIN is required for proper formation and constriction of the septal actomyosin ring. However, MST-1 also interacts with several components of the MOR network and modulates MOR activity at multiple levels. MST-1 functions as promiscuous enzyme and also activates the MOR effector kinase COT-1 through hydrophobic motif phosphorylation. In addition, MST-1 physically interacts with the MOR kinase POD-6, and dimerization of both proteins inactivates the GC kinase hetero-complex. These data specify an antagonistic relationship between the SIN and MOR during septum formation in the filamentous ascomycete model Neurospora crassa that is, at least in part, coordinated through the GC kinase MST-1. The similarity of the SIN and MOR pathways to the animal Hippo and Ndr pathways, respectively, suggests that intensive cross-communication between distinct NDR kinase modules may also be relevant for the homologous NDR kinases of higher eukaryotes. PMID:24762679

  6. Molecular determinants for cardiovascular TRPC6 channel regulation by Ca2+/calmodulin-dependent kinase II

    DEFF Research Database (Denmark)

    Shi, Juan; Geshi, Naomi; Takahashi, Shinichi;

    2013-01-01

    The molecular mechanism underlying Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII)-mediated regulation of the mouse transient receptor potential channel TRPC6 was explored by chimera, deletion and site-directed mutagenesis approaches. Induction of currents (ICCh) in TRPC6-expressing HEK293 cel...... essential for CaMKII-mediated regulation of TRPC6 channels. This mechanism may be of physiological significance in a native environment such as in vascular smooth muscle cells....

  7. Identification of a BET family Bromodomain / Casein Kinase II / TAF-containing complex as a regulator of mitotic condensin function

    OpenAIRE

    Kim, Hyun-Soo; Mukhopadhyay, Rituparna; Rothbart, Scott B.; Silva, Andrea C.; Vanoosthuyse, Vincent; Radovani, Ernest; Kislinger, Thomas; Roguev, Assen; Ryan, Colm J.; Xu, Jiewei; Jahari, Harlizawati; Hardwick, Kevin G.; Greenblatt, Jack F.; Krogan, Nevan J.; Fillingham, Jeffrey S.

    2014-01-01

    Condensin is a central regulator of mitotic genome structure, with mutants showing poorly condensed chromosomes and profound segregation defects. Here we identify NCT complex, comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), Casein Kinase II (CKII) and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions, but only briefly co-localize during the periods of chromosome condensation and decondensation. This pattern ...

  8. Dendritic spine changes in the development of alcohol addiction regulated by α-calcium/calmodulin-dependent protein kinase II

    OpenAIRE

    Zofia Mijakowska

    2014-01-01

    Introduction Alcohol has many adverse effects on the brain. Among them are dendritic spine morphology alterations, which are believed to be the basis of alcohol addiction. Autophosphorylation of α-calcium/calmodulin-dependent protein kinase II (αCaMKII) has been shown to regulate spine morphology in vitro. Here we show that αCaMKII can also regulate addiction related behaviour and dendritic spine morphology changes caused by alcohol consumption in vivo. Method 12 αCaMKII-autophosphorylatio...

  9. Rho GTPase/Rho Kinase Negatively Regulates Endothelial Nitric Oxide Synthase Phosphorylation through the Inhibition of Protein Kinase B/Akt in Human Endothelial Cells

    Science.gov (United States)

    Ming, Xiu-Fen; Viswambharan, Hema; Barandier, Christine; Ruffieux, Jean; Kaibuchi, Kozo; Rusconi, Sandro; Yang, Zhihong

    2002-01-01

    Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB. PMID:12446767

  10. Expression and Characterization of ArgR, An Arginine Regulatory Protein in Corynebacterium crenatum

    Institute of Scientific and Technical Information of China (English)

    CHEN Xue Lan; ZHANG Bin; TANG Li; JIAO Hai Tao; XU Heng Yi; XU Feng; XU Hong; WEI Hua; XIONG Yong Hua

    2014-01-01

    Objective Corynebacterium crenatum MT, a mutant from C. crenatum AS 1.542 with a lethal argR gene, exhibits high arginine production. To confirm the effect of ArgR on arginine biosynthesis in C. crenatum, an intact argR gene from wild-type AS 1.542 was introduced into C. crenatum MT, resulting in C. crenatum MT. sp, and the changes of transcriptional levels of the arginine biosynthetic genes and arginine production were compared between the mutant strain and the recombinant strain. Methods Quantitative real-time polymerase chain reaction was employed to analyze the changes of the related genes at the transcriptional level, electrophoretic mobility shift assays were used to determine ArgR binding with the argCJBDF, argGH, and carAB promoter regions, and arginine production was determined with an automated amino acid analyzer. Results Arginine production assays showed a 69.9%reduction in arginine from 9.01±0.22 mg/mL in C. crenatum MT to 2.71±0.13 mg/mL (P Conclusion The arginine biosynthetic genes in C. crenatum are clearly controlled by the negative regulator ArgR, and intact ArgR in C. crenatum MT results in a significant descrease in arginine production.

  11. Energy-sensitive regulation of Na+/K+-ATPase by Janus kinase 2.

    Science.gov (United States)

    Bhavsar, Shefalee K; Hosseinzadeh, Zohreh; Brenner, Dirk; Honisch, Sabina; Jilani, Kashif; Liu, Guoxing; Szteyn, Kalina; Sopjani, Mentor; Mak, Tak W; Shumilina, Ekaterina; Lang, Florian

    2014-02-15

    Janus kinase 2 (JAK2) contributes to intracellular signaling of leptin and erythropoietin, hormones protecting cells during energy depletion. The present study explores whether JAK2 is activated by energy depletion and regulates Na(+)/K(+)-ATPase, the major energy-consuming pump. In Jurkat cells, JAK2 activity was determined by radioactive kinase assay, phosphorylated JAK2 detected by Western blotting, ATP levels measured by luciferase assay, as well as Na(+)/K(+)-ATPase α1-subunit transcript and protein abundance determined by real-time PCR and Western blotting, respectively. Ouabain-sensitive K(+)-induced currents (Ipump) were measured by whole cell patch clamp. Ipump was further determined by dual-electrode voltage clamp in Xenopus oocytes injected with cRNA-encoding JAK2, active (V617F)JAK2, or inactive (K882E)JAK2. As a result, in Jurkat T cells, JAK2 activity significantly increased following energy depletion by sodium azide (NaN3) or 2,4- dinitro phenol (DNP). DNP- and NaN3-induced decrease of cellular ATP was significantly augmented by JAK2 inhibitor AG490 and blunted by Na(+)/K(+)-ATPase inhibitor ouabain. DNP decreased and AG490 enhanced Ipump as well as Na(+)/K(+)-ATPase α1-subunit transcript and protein abundance. The α1-subunit transcript levels were also enhanced by signal transducer and activator of transcription-5 inhibitor CAS 285986-31-4. In Xenopus oocytes, Ipump was significantly decreased by expression of JAK2 and (V617F)JAK2 but not of (K882E)JAK2, effects again reversed by AG490. In (V617F)JAK2-expressing Xenopus oocytes, neither DNP nor NaN3 resulted in further decline of Ipump. In Xenopus oocytes, the effect of (V617F)JAK2 on Ipump was not prevented by inhibition of transcription with actinomycin. In conclusion, JAK2 is a novel energy-sensing kinase that curtails energy consumption by downregulating Na(+)/K(+)-ATPase expression and activity. PMID:24304834

  12. Molecular characterization of a signal-regulated kinase homolog from Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    LI Jing; ZHANG Chuan-shan; L(U) Guo-dong; WANG Jun-hua; WEN Hao; YAN Gen-qiang; WEI Xu-fa; LIN Ren-yong

    2011-01-01

    Background Cystic echinococcosis due to Echinococcus granulosus (E. granulosus) is one of the most important chronic helminthic diseases, especially in sheep/cattle-raising regions. The larval stage of the parasite forms a cyst that grows in the liver, lung, or other organs ofthe host. To ensure a long life in the host tissues, the parasite establishes complex inter-cellular communication systems between its host to allow its differentiation toward each larval stage.Recent studies have reported that this communication is associated with the extracellular signal-regulated kinase (ERK)mitogen-activated protein kinase cascade in helminth parasites, and in particular that these protein kinases might serve as effective targets for a novel chemotherapy for cystic echinococcosis. The aim of the present study investigated the biological function of a novel ERK ortholog from E. granulosus, EgERK.Methods DNA encoding EgERK was isolated from protoscolices of E. granulosus and analyzed using the LA Taq polymerase chain reaction (PCR) approach and bioinformatics. Reverse transcription PCR (RT-PCR) was used to determine the transcription level of the gene at two different larval tissues. Western blotting was used to detect levels of EgERK protein. The expression profile of EgERK in protoscolices was examined by immunofluorescence.Results We cloned the entire Egerk genomic locus from E. granulosus. In addition, two alternatively spliced transcripts of Egerk, Egerk-A, and Egerk-B were identified. Egerk-A was found to constitutively expressed at the transcriptional and protein levels in two different larval tissues (cyst membranes and protoscolices). Egerk-A was expressed in the tegumental structures, hooklets, and suckers and in the tissue surrounding the rostellum of E. granulosus protoscolices.Conclusions We have cloned the genomic DNA of a novel ERK ortholog from E. granulosus, EgERK (GenBank ID HQ585923), and found that it is constitutively expressed in cyst membrane and

  13. Circadian metabolic regulation through crosstalk between casein kinase 1δ and transcriptional coactivator PGC-1α.

    Science.gov (United States)

    Li, Siming; Chen, Xiao-Wei; Yu, Lei; Saltiel, Alan R; Lin, Jiandie D

    2011-12-01

    Circadian clock coordinates behavior and physiology in mammals in response to light and feeding cycles. Disruption of normal clock function is associated with increased risk for cardiovascular and metabolic diseases, underscoring the emerging concept that temporal regulation of tissue metabolism is a fundamental aspect of energy homeostasis. We have previously demonstrated that transcriptional coactivator, peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), coordinates circadian metabolic rhythms through simultaneous regulation of metabolic and clock gene expression. In this study, we found that PGC-1α physically interacts with, and is phosphorylated by, casein kinase 1δ (CK1δ), a core component of the circadian pacemaker. CK1δ represses the transcriptional function of PGC-1α in cultured hepatocytes, resulting in decreased gluconeogenic gene expression and glucose secretion. At the molecular level, CK1δ phosphorylation of PGC-1α within its arginine/serine-rich domain enhances its degradation through the proteasome system. Together, these results elucidate a novel mechanism through which circadian pacemaker transduces timing signals to the metabolic regulatory network that controls hepatic energy metabolism.

  14. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Kovács, Krisztián A.

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  15. Protein kinase C regulates the phosphorylation and oligomerization of ERM binding phosphoprotein 50

    International Nuclear Information System (INIS)

    Ezrin-Radixin-Moesin (ERM) binding phosphoprotein 50 (EBP50, a.k.a. NHERF-1) is a scaffold protein essential for the localization and coordinated activity of apical transporters, enzymes and receptors in epithelial cells. EBP50 acts via multiple protein binding interactions, including oligomerization through interactions of its PSD95-Dlg-ZO1 (PDZ) domains. EBP50 can be phosphorylated on multiple sites and phosphorylation of specific sites modulates the extent of oligomerization. The aim of the present study was to test the capacity of protein kinase C (PKC) to phosphorylate EBP50 and to regulate its oligomerization. In vitro experiments showed that the catalytic subunit of PKC directly phosphorylates EBP50. In HEK-293 cells transfected with rat EBP50 cDNA, a treatment with 12 myristate 13-acetate (PMA) induced a translocation of PKCα and β isoforms to the membrane and increased 32P incorporation into EBP50. In co-transfection/co-precipitation studies, PMA treatment stimulated EBP50 oligomerization. Mass spectrometry analysis of full-length EBP50 and phosphorylation analyses of specific domains, and of mutated or truncated forms of EBP50, indicated that PKC-induced phosphorylation of EBP50 occurred on the Ser337/Ser338 residue within the carboxyl-tail domain of the protein. Truncation of Ser337/Ser338 also diminished PKC-induced oligomerization of EBP50. These results suggest the PKC signaling pathway can impact EBP50-dependent cellular functions by regulating EBP50 oligomerization

  16. Learned stressor resistance requires extracellular signal-regulated kinase in the prefrontal cortex

    Directory of Open Access Journals (Sweden)

    John Paul Christianson

    2014-10-01

    Full Text Available Behaviorally controllable stressors confer protection from the neurochemical and behavioral consequences of future uncontrollable stressors, a phenomenon termed behavioral immunization. Recent data implicate neuroplasticity within the ventromedial prefrontal cortex (mPFC as critical to behavioral immunization. Adult, male Sprague-Dawley rats were exposed to a series of controllable tailshocks and one week later to uncontrollable tailshocks, followed 24h later by social exploration and shuttlebox escape tests. To test the involvement of N-methyl-D-aspartate receptors (NMDAR and the extracellular signal-regulated kinase (ERK cascade in behavioral immunization, either D-AP5 or the MEK inhibitor U0126 was injected to the prelimbic (PL or infralimbic (IL mPFC prior to controllable stress exposure. Phosphorylated ERK and P70S6K, regulators of transcription and translation, were quantified by Western blot or immunohistochemistry after controllable or uncontrollable tailshocks. Prior controllable stress prevented the social exploration and shuttlebox performance deficits caused by the later uncontrollable stressor, and this effect was blocked by injections of D-AP5 into mPFC. A significant increase in phosphorylated ERK1 and ERK2, but not P70S6K, occurred within the PL and IL in rats exposed to controllable stress, but not to uncontrollable stress. However, U0126 only prevented behavioral immunization when injected to the PL. We provide evidence that NMDAR and ERK dependent plasticity within the PL region is required for behavioral immunization, a learned form of stressor resistance.

  17. Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesis.

    Science.gov (United States)

    Ferreira, Tânia; Prudêncio, Pedro; Martinho, Rui Gonçalo

    2014-10-15

    Nurse cell dumping is an actin-myosin based process, where 15 nurse cells of a given egg chamber contract and transfer their cytoplasmic content through the ring canals into the growing oocyte. We isolated two mutant alleles of protein kinase N (pkn) and showed that Pkn negatively-regulates activation of the actin-myosin cytoskeleton during the onset of dumping. Using live-cell imaging analysis we observed that nurse cell dumping rates sharply increase during the onset of fast dumping. Such rate increase was severely impaired in pkn mutant nurse cells due to excessive nurse cell actin-myosin activity and/or loss of tissue integrity. Our work demonstrates that the transition between slow and fast dumping is a discrete event, with at least a five to six-fold dumping rate increase. We show that Pkn negatively regulates nurse cell actin-myosin activity. This is likely to be important for directional cytoplasmic flow. We propose Pkn provides a negative feedback loop to help avoid excessive contractility after local activation of Rho GTPase.

  18. Computational Modeling of the Metabolic States Regulated by the Kinase Akt

    Science.gov (United States)

    Mosca, Ettore; Alfieri, Roberta; Maj, Carlo; Bevilacqua, Annamaria; Canti, Gianfranco; Milanesi, Luciano

    2012-01-01

    Signal transduction and gene regulation determine a major reorganization of metabolic activities in order to support cell proliferation. Protein Kinase B (PKB), also known as Akt, participates in the PI3K/Akt/mTOR pathway, a master regulator of aerobic glycolysis and cellular biosynthesis, two activities shown by both normal and cancer proliferating cells. Not surprisingly considering its relevance for cellular metabolism, Akt/PKB is often found hyperactive in cancer cells. In the last decade, many efforts have been made to improve the understanding of the control of glucose metabolism and the identification of a therapeutic window between proliferating cancer cells and proliferating normal cells. In this context, we have modeled the link between the PI3K/Akt/mTOR pathway, glycolysis, lactic acid production, and nucleotide biosynthesis. We used a computational model to compare two metabolic states generated by two different levels of signaling through the PI3K/Akt/mTOR pathway: one of the two states represents the metabolism of a growing cancer cell characterized by aerobic glycolysis and cellular biosynthesis, while the other state represents the same metabolic network with a reduced glycolytic rate and a higher mitochondrial pyruvate metabolism. Biochemical reactions that link glycolysis and pentose phosphate pathway revealed their importance for controlling the dynamics of cancer glucose metabolism. PMID:23181020

  19. 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

  20. Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers

    Science.gov (United States)

    Poovaiah, B. W.; Xia, M.; Liu, Z.; Wang, W.; Yang, T.; Sathyanarayanan, P. V.; Franceschi, V. R.

    1999-01-01

    Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca(2+) and Ca(2+)/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the beta-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by beta-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca(2+) concentration in target cells, thereby controlling developmental events in the anther.

  1. RAF kinase activity regulates neuroepithelial cell proliferation and neuronal progenitor cell differentiation during early inner ear development.

    Directory of Open Access Journals (Sweden)

    Marta Magariños

    Full Text Available BACKGROUND: Early inner ear development requires the strict regulation of cell proliferation, survival, migration and differentiation, coordinated by the concerted action of extrinsic and intrinsic factors. Deregulation of these processes is associated with embryonic malformations and deafness. We have shown that insulin-like growth factor I (IGF-I plays a key role in embryonic and postnatal otic development by triggering the activation of intracellular lipid and protein kinases. RAF kinases are serine/threonine kinases that regulate the highly conserved RAS-RAF-MEK-ERK signaling cascade involved in transducing the signals from extracellular growth factors to the nucleus. However, the regulation of RAF kinase activity by growth factors during development is complex and still not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: By using a combination of qRT-PCR, Western blotting, immunohistochemistry and in situ hybridization, we show that C-RAF and B-RAF are expressed during the early development of the chicken inner ear in specific spatiotemporal patterns. Moreover, later in development B-RAF expression is associated to hair cells in the sensory patches. Experiments in ex vivo cultures of otic vesicle explants demonstrate that the influence of IGF-I on proliferation but not survival depends on RAF kinase activating the MEK-ERK phosphorylation cascade. With the specific RAF inhibitor Sorafenib, we show that blocking RAF activity in organotypic cultures increases apoptosis and diminishes the rate of cell proliferation in the otic epithelia, as well as severely impairing neurogenesis of the acoustic-vestibular ganglion (AVG and neuron maturation. CONCLUSIONS/SIGNIFICANCE: We conclude that RAF kinase activity is essential to establish the balance between cell proliferation and death in neuroepithelial otic precursors, and for otic neuron differentiation and axonal growth at the AVG.

  2. Dopamine D1 Receptors Regulate Protein Synthesis-Dependent Long-Term Recognition Memory via Extracellular Signal-Regulated Kinase 1/2 in the Prefrontal Cortex

    Science.gov (United States)

    Nagai, Taku; Takuma, Kazuhiro; Kamei, Hiroyuki; Ito, Yukio; Nakamichi, Noritaka; Ibi, Daisuke; Nakanishi, Yutaka; Murai, Masaaki; Mizoguchi, Hiroyuki; Nabeshima, Toshitaka; Yamada, Kiyofumi

    2007-01-01

    Several lines of evidence suggest that extracellular signal-regulated kinase1/2 (ERK1/2) and dopaminergic system is involved in learning and memory. However, it remains to be determined if the dopaminergic system and ERK1/2 pathway contribute to cognitive function in the prefrontal cortex (PFC). The amount of phosphorylated ERK1/2 was increased in…

  3. Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Qiao, Lan [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Pharmaceutical Sciences, Jilin University, Changchun 130021 (China); Paul, Pritha; Lee, Sora [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Qiao, Jingbo [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Wang, Yongsheng [Department of Pharmaceutical Sciences, Jilin University, Changchun 130021 (China); Chung, Dai H., E-mail: dai.chung@vanderbilt.edu [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States)

    2013-05-31

    Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated that GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma.

  4. Quantitative phosphoproteomics of protein kinase SnRK1 regulated protein phosphorylation in Arabidopsis under submergence.

    Science.gov (United States)

    Cho, Hsing-Yi; Wen, Tuan-Nan; Wang, Ying-Tsui; Shih, Ming-Che

    2016-04-01

    SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of the plant stress and energy starvation signalling pathways. We observed that the Arabidopsis SnRK1.1 dominant negative mutant (SnRK1.1 (K48M) ) had lower tolerance to submergence than the wild type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in signalling pathways triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5-3h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1 (K48M) under submergence. These proteins are involved mainly in sugar and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under abiotic stresses, was disrupted in the SnRK1.1 (K48M) mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1. We also showed that energy conservation was disrupted in SnRK1.1 (K48M) , mpk6, and PTP1 (S7AS8A) under submergence. These results reveal insights into the function of SnRK1 and the downstream signalling factors related to submergence. PMID:27029354

  5. Protein kinase C regulates tonic GABAA receptor-mediated inhibition in the hippocampus and thalamus

    Science.gov (United States)

    Bright, Damian P; Smart, Trevor G

    2013-01-01

    Tonic inhibition mediated by extrasynaptic GABAA receptors (GABAARs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABAARs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole-cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC-mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABAAR-mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABAAR activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABAARs, which represent a key extrasynaptic GABAAR isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABAARs. The inhibitory effects of PKC activation on α4β2δ GABAAR activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC-mediated phosphorylation can be an important physiological regulator of tonic GABAAR-mediated inhibition. PMID:24102973

  6. Mining frequent patterns for AMP-activated protein kinase regulation on skeletal muscle

    Directory of Open Access Journals (Sweden)

    Chen Yi-Ping

    2006-08-01

    Full Text Available Abstract Background AMP-activated protein kinase (AMPK has emerged as a significant signaling intermediary that regulates metabolisms in response to energy demand and supply. An investigation into the degree of activation and deactivation of AMPK subunits under exercise can provide valuable data for understanding AMPK. In particular, the effect of AMPK on muscle cellular energy status makes this protein a promising pharmacological target for disease treatment. As more AMPK regulation data are accumulated, data mining techniques can play an important role in identifying frequent patterns in the data. Association rule mining, which is commonly used in market basket analysis, can be applied to AMPK regulation. Results This paper proposes a framework that can identify the potential correlation, either between the state of isoforms of α, β and γ subunits of AMPK, or between stimulus factors and the state of isoforms. Our approach is to apply item constraints in the closed interpretation to the itemset generation so that a threshold is specified in terms of the amount of results, rather than a fixed threshold value for all itemsets of all sizes. The derived rules from experiments are roughly analyzed. It is found that most of the extracted association rules have biological meaning and some of them were previously unknown. They indicate direction for further research. Conclusion Our findings indicate that AMPK has a great impact on most metabolic actions that are related to energy demand and supply. Those actions are adjusted via its subunit isoforms under specific physical training. Thus, there are strong co-relationships between AMPK subunit isoforms and exercises. Furthermore, the subunit isoforms are correlated with each other in some cases. The methods developed here could be used when predicting these essential relationships and enable an understanding of the functions and metabolic pathways regarding AMPK.

  7. Protein kinase C-dependent regulation of human hepatic drug transporter expression.

    Science.gov (United States)

    Mayati, Abdullah; Le Vee, Marc; Moreau, Amélie; Jouan, Elodie; Bucher, Simon; Stieger, Bruno; Denizot, Claire; Parmentier, Yannick; Fardel, Olivier

    2015-12-15

    Hepatic drug transporters are now recognized as major actors of hepatobiliary elimination of drugs. Characterization of their regulatory pathways is therefore an important issue. In this context, the present study was designed to analyze the potential regulation of human hepatic transporter expression by protein kinase C (PKC) activation. Treatment by the reference PKC activator phorbol 12-myristate 13-acetate (PMA) for 48h was shown to decrease mRNA expression of various sinusoidal transporters, including OATP1B1, OATP2B1, NTCP, OCT1 and MRP3, but to increase that of OATP1B3, whereas mRNA expression of canalicular transporters was transiently enhanced (MDR1), decreased (BSEP and MRP2) or unchanged (BCRP) in human hepatoma HepaRG cells. The profile of hepatic transporter mRNA expression changes in PMA-treated HepaRG cells was correlated to that found in PMA-exposed primary human hepatocytes and was similarly observed in response to the PKC-activating marketed drug ingenol mebutate. It was associated with concomitant repression of OATP1B1 and OATP2B1 protein expression and reduction of OATP, OCT1, NTCP and MRP2 activity. The use of chemical PKC inhibitors further suggested a contribution of novel PKCs isoforms to PMA-mediated regulations of transporter mRNA expression. PMA was finally shown to cause epithelial-mesenchymal transition (EMT) in HepaRG cells and exposure to various additional EMT inducers, i.e., hepatocyte growth factor, tumor growth factor-β1 or the HNF4α inhibitor BI6015, led to transporter expression alterations highly correlated to those triggered by PMA. Taken together, these data highlight PKC-dependent regulation of human hepatic drug transporter expression, which may be closely linked to EMT triggered by PKC activation. PMID:26462574

  8. Regulation of post-translational modifications of muskelin by protein kinase C.

    Science.gov (United States)

    Prag, Soren; De Arcangelis, Adèle; Georges-Labouesse, Elisabeth; Adams, Josephine C

    2007-01-01

    Muskelin is a member of the kelch-repeat superfamily of proteins, identified as an intracellular protein involved in cell spreading responses to thombospondin-1. Muskelin is expressed by many adult tissues and has an evolutionarily conserved, multidomain architecture consisting of an amino-terminal discoidin-like domain, a central alpha-helical region and six kelch-repeats that are predicted to form a beta-propeller structure. We previous demonstrated that muskelin molecules undergo head-to-tail association, however the physiological, post-translational regulation of muskelin is not well understood. Here, we have examined the expression of muskelin during mouse embryonic development and report widespread expression that includes muscle tissues, multiple epithelia and the brain. In cultured skeletal myoblasts and vascular smooth muscle cells, muskelin exists as a complex set of isoelectric variants. Five potential sites for phosphorylation by protein kinase C (PKC), are conserved between vertebrate and Drosophila muskelins, therefore we examined the hypothesis that muskelin is regulated post-translationally by PKC activity. We demonstrate that PKC activation or inhibition regulates the profile of endogenous muskelin isoelectric variants and that muskelin is a substrate for PKCalphain vitro. Wild-type GFP-muskelin and a panel of alanine point mutations were used to test the sensitivity of self-association to PKC activation. Mutation of two of the sites, S324 and T515, partially inhibited the ability of muskelin to self-associate in cells and inhibited responsiveness to activated PKC. Interestingly, both sites are predicted to lie in surface-exposed loops on the same side of the beta-propeller, implicating a common binding interface. PMID:17049906

  9. AMP-activated protein kinase (AMPK) activation regulates in vitro bone formation and bone mass.

    Science.gov (United States)

    Shah, M; Kola, B; Bataveljic, A; Arnett, T R; Viollet, B; Saxon, L; Korbonits, M; Chenu, C

    2010-08-01

    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 cultured in the presence of AMPK activators (AICAR and metformin), AMPK inhibitor (compound C), the gastric peptide hormone ghrelin and the beta-adrenergic blocker propranolol. AMPK activity was measured in cell lysates by a functional kinase assay and AMPK protein phosphorylation was studied by Western Blotting using an antibody recognizing AMPK Thr-172 residue. We demonstrated that treatment of ROS 17/2.8 cells with AICAR and metformin stimulates Thr-172 phosphorylation of AMPK and dose-dependently increases its activity. In contrast, treatment of ROS 17/2.8 cells with compound C inhibited AMPK phosphorylation. Ghrelin and propranolol dose-dependently increased AMPK phosphorylation and activity. Cell proliferation and alkaline phosphatase activity were not affected by metformin treatment while AICAR significantly inhibited ROS 17/2.8 cell proliferation and alkaline phosphatase activity at high concentrations. To study the effect of AMPK activation on bone formation in vitro, primary osteoblasts obtained from rat calvaria were cultured for 14-17days in the presence of AICAR, metformin and compound C. Formation of 'trabecular-shaped' bone nodules was evaluated following alizarin red staining. We demonstrated that both AICAR and metformin dose-dependently increase trabecular bone nodule formation, while compound C inhibits bone formation. When primary osteoblasts were co-treated with AICAR and compound C, compound C suppressed the stimulatory effect of AICAR on bone nodule formation

  10. Sphingosine kinase-1 is central to androgen-regulated prostate cancer growth and survival.

    Directory of Open Access Journals (Sweden)

    Audrey Dayon

    Full Text Available BACKGROUND: Sphingosine kinase-1 (SphK1 is an oncogenic lipid kinase notably involved in response to anticancer therapies in prostate cancer. Androgens regulate prostate cancer cell proliferation, and androgen deprivation therapy is the standard of care in the management of patients with advanced disease. Here, we explored the role of SphK1 in the regulation of androgen-dependent prostate cancer cell growth and survival. METHODOLOGY/PRINCIPAL FINDINGS: Short-term androgen removal induced a rapid and transient SphK1 inhibition associated with a reduced cell growth in vitro and in vivo, an event that was not observed in the hormono-insensitive PC-3 cells. Supporting the critical role of SphK1 inhibition in the rapid effect of androgen depletion, its overexpression could impair the cell growth decrease. Similarly, the addition of dihydrotestosterone (DHT to androgen-deprived LNCaP cells re-established cell proliferation, through an androgen receptor/PI3K/Akt dependent stimulation of SphK1, and inhibition of SphK1 could markedly impede the effects of DHT. Conversely, long-term removal of androgen support in LNCaP and C4-2B cells resulted in a progressive increase in SphK1 expression and activity throughout the progression to androgen-independence state, which was characterized by the acquisition of a neuroendocrine (NE-like cell phenotype. Importantly, inhibition of the PI3K/Akt pathway--by negatively impacting SphK1 activity--could prevent NE differentiation in both cell models, an event that could be mimicked by SphK1 inhibitors. Fascinatingly, the reversability of the NE phenotype by exposure to normal medium was linked with a pronounced inhibition of SphK1 activity. CONCLUSIONS/SIGNIFICANCE: We report the first evidence that androgen deprivation induces a differential effect on SphK1 activity in hormone-sensitive prostate cancer cell models. These results also suggest that SphK1 activation upon chronic androgen deprivation may serve as a

  11. Dihydrotestosterone regulating apolipoprotein M expression mediates via protein kinase C in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Yi-zhou Ye

    2012-12-01

    Full Text Available Abstract Background Administration of androgens decreases plasma concentrations of high-density lipid cholesterol (HDL-C. However, the mechanisms by which androgens mediate lipid metabolism remain unknown. This present study used HepG2 cell cultures and ovariectomized C57BL/6 J mice to determine whether apolipoprotein M (ApoM, a constituent of HDL, was affected by dihydrotestosterone (DHT. Methods HepG2 cells were cultured in the presence of either DHT, agonist of protein kinase C (PKC, phorbol-12-myristate-13-acetate (PMA, blocker of androgen receptor flutamide together with different concentrations of DHT, or DHT together with staurosporine at different concentrations for 24 hrs. Ovariectomized C57BL/6 J mice were treated with DHT or vehicle for 7d or 14d and the levels of plasma ApoM and livers ApoM mRNA were measured. The mRNA levels of ApoM, ApoAI were determined by real-time RT-PCR. ApoM and ApoAI were determined by western blotting analysis. Results Addition of DHT to cell culture medium selectively down-regulated ApoM mRNA expression and ApoM secretion in a dose-dependent manner. At 10 nM DHT, the ApoM mRNA levels were about 20% lower than in untreated cells and about 40% lower at 1000 nM DHT than in the control cells. The secretion of ApoM into the medium was reduced to a similar extent. The inhibitory effect of DHT on ApoM secretion was not blocked by the classical androgen receptor blocker flutamide but by an antagonist of PKC, Staurosporine. Agonist of PKC, PMA, also reduced ApoM. At 0.5 μM PMA, the ApoM mRNA levels and the secretion of ApoM into the medium were about 30% lower than in the control cells. The mRNA expression levels and secretion of another HDL-associated apolipoprotein AI (ApoAI were not affected by DHT. The levels of plasma ApoM and liver ApoM mRNA of DHT-treated C57BL/6 J mice were lower than those of vehicle-treated mice. Conclusions DHT directly and selectively down-regulated the level of ApoM mRNA and the

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-03-01

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

  13. Molecular physiology of SPAK and OSR1: two Ste20-related protein kinases regulating ion transport.

    Science.gov (United States)

    Gagnon, Kenneth B; Delpire, Eric

    2012-10-01

    SPAK (Ste20-related proline alanine rich kinase) and OSR1 (oxidative stress responsive kinase) are members of the germinal center kinase VI subfamily of the mammalian Ste20 (Sterile20)-related protein kinase family. Although there are 30 enzymes in this protein kinase family, their conservation across the fungi, plant, and animal kingdom confirms their evolutionary importance. Already, a large volume of work has accumulated on the tissue distribution, binding partners, signaling cascades, and physiological roles of mammalian SPAK and OSR1 in multiple organ systems. After reviewing this basic information, we will examine newer studies that demonstrate the pathophysiological consequences to SPAK and/or OSR1 disruption, discuss the development and analysis of genetically engineered mouse models, and address the possible role these serine/threonine kinases might have in cancer proliferation and migration. PMID:23073627

  14. The human Na(+)/H(+) exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

    DEFF Research Database (Denmark)

    Hendus-Altenburger, Ruth; Pedraz-Cuesta, Elena; Olesen, Christina W;

    2016-01-01

    BACKGROUND: Extracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding. METHODS AND RESULTS: Here, we identify...... the human Na(+)/H(+) exchanger 1 (hNHE1) as a membrane scaffold protein for ERK2 and show direct hNHE1-ERK1/2 interaction in cellular contexts. Using nuclear magnetic resonance (NMR) spectroscopy and immunofluorescence analysis we demonstrate that ERK2 scaffolding by hNHE1 occurs by one of three D...... and ERK2, and provides a molecular mechanism for the important ERK2 scaffolding function of the membrane protein hNHE1, which regulates the phosphorylation of both hNHE1 and ERK2....

  15. Phosphorylation and inactivation of glycogen synthase kinase 3β (GSK3β) by dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A).

    Science.gov (United States)

    Song, Woo-Joo; Song, Eun-Ah Christine; Jung, Min-Su; Choi, Sun-Hee; Baik, Hyung-Hwan; Jin, Byung Kwan; Kim, Jeong Hee; Chung, Sul-Hee

    2015-01-23

    Glycogen synthase kinase 3β (GSK3β) participates in many cellular processes, and its dysregulation has been implicated in a wide range of diseases such as obesity, type 2 diabetes, cancer, and Alzheimer disease. Inactivation of GSK3β by phosphorylation at specific residues is a primary mechanism by which this constitutively active kinase is controlled. However, the regulatory mechanism of GSK3β is not fully understood. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) has multiple biological functions that occur as the result of phosphorylation of diverse proteins that are involved in metabolism, synaptic function, and neurodegeneration. Here we show that GSK3β directly interacts with and is phosphorylated by Dyrk1A. Dyrk1A-mediated phosphorylation at the Thr(356) residue inhibits GSK3β activity. Dyrk1A transgenic (TG) mice are lean and resistant to diet-induced obesity because of reduced fat mass, which shows an inverse correlation with the effect of GSK3β on obesity. This result suggests a potential in vivo association between GSK3β and Dyrk1A regarding the mechanism underlying obesity. The level of Thr(P)(356)-GSK3β was higher in the white adipose tissue of Dyrk1A TG mice compared with control mice. GSK3β activity was differentially regulated by phosphorylation at different sites in adipose tissue depending on the type of diet the mice were fed. Furthermore, overexpression of Dyrk1A suppressed the expression of adipogenic proteins, including peroxisome proliferator-activated receptor γ, in 3T3-L1 cells and in young Dyrk1A TG mice fed a chow diet. Taken together, these results reveal a novel regulatory mechanism for GSK3β activity and indicate that overexpression of Dyrk1A may contribute to the obesity-resistant phenotype through phosphorylation and inactivation of GSK3β. PMID:25477508

  16. Directed mutagenesis of deoxyguanosine site at arginine 79 up-regulates turnover on deoxyadenosine kinase subunit of heterodimeric enzyme from Lactobacillus acidophilus R26.

    Science.gov (United States)

    Hong, Y S; Ma, G T; Ives, D H

    1995-03-24

    Examination of conserved motifs on the cloned subunits of the deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) of Lactobacillus acidophilus R-26 has begun with the Asp-Arg-Ser (DRS) motif. Replacement of Asp-78 of both subunits with Glu, Ala, or Asn reduced dGK and dAK activities to less than 0.2%, whereas replacement of Arg-79 with Lys, either on both subunits in tandem (R79K), or on the dGK subunit only (R79K:dGK), yielded active but kinetically modified enzymes. These were partially purified, and their kinetic and regulatory properties were analyzed. For dAK activity, the Vmax of the R79K:dGK enzyme was increased 28-fold, with no change in the limiting Km for dAdo, but with a slightly reduced Km for MgATP. The V/K efficiency ratio of dAK was also increased 29-fold, but that of dGK was decreased to 5-10% due to a 10-fold increase in Km for dGuo and a reduced Vmax. Therefore, the R79K substitution seems to have a greater effect on dGuo binding than on that of dAdo, but dGK modification appears to produce a stimulatory conformational effect on the opposite subunit, resembling the known unidirectional activation of dAK by either dGuo or dGTP.

  17. E2~Ub conjugates regulate the kinase activity of Shigella effector OspG during pathogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Pruneda, Jonathan N. [Department of Biochemistry, University of Washington, Seattle WA USA; Smith, F. Donelson [Howard Hughes Medical Institute, Department of Pharmacology, University of Washington, Seattle WA USA; Daurie, Angela [Department of Microbiology and Immunology, Dalhousie University, Halifax NS Canada; Swaney, Danielle L. [Department of Genome Sciences, University of Washington, Seattle WA USA; Villén, Judit [Department of Genome Sciences, University of Washington, Seattle WA USA; Scott, John D. [Howard Hughes Medical Institute, Department of Pharmacology, University of Washington, Seattle WA USA; Stadnyk, Andrew W. [Department of Pediatrics, Dalhousie University, Halifax NS Canada; Le Trong, Isolde [Department of Biological Structure, University of Washington, Seattle WA USA; Stenkamp, Ronald E. [Department of Biochemistry, University of Washington, Seattle WA USA; Department of Biological Structure, University of Washington, Seattle WA USA; Klevit, Rachel E. [Department of Biochemistry, University of Washington, Seattle WA USA; Rohde, John R. [Department of Microbiology and Immunology, Dalhousie University, Halifax NS Canada; Brzovic, Peter S. [Department of Biochemistry, University of Washington, Seattle WA USA

    2014-01-20

    Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin-conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A cocrystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.

  18. Mitogen-activated protein kinases (p38 and c-Jun NH2-terminal kinase) are differentially regulated during cardiac volume and pressure overload hypertrophy.

    Science.gov (United States)

    Sopontammarak, Somkiat; Aliharoob, Assad; Ocampo, Catherina; Arcilla, Rene A; Gupta, Mahesh P; Gupta, Madhu

    2005-01-01

    Chronic pressure overload (PO) and volume overload (VO) result in morphologically and functionally distinct forms of myocardial hypertrophy. However, the molecular mechanism initiating these two types of hypertrophy is not yet understood. Data obtained from different cell types have indicated that the mitogen-activated protein kinases (MAPKs) comprising c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 play an important role in transmitting signals of stress stimuli to elicit the cellular response. We tested the hypothesis that early induction of MAPKs differs in two types of overload on the heart and associates with distinct expression of hypertrophic marker genes, namely ANF, alpha-myosin heavy chain (alpha-MHC), and beta-MHC. In rats, VO was induced by aortocaval shunt and PO by constriction of the abdominal aorta. The PO animals were further divided into two groups depending on the severity of the constriction, mild (MPO) and severe pressure overload (SPO), having 35 and 85% aortic constriction, respectively. Early changes in MAPK activity (2-120 min and 1 to 2 d) were analyzed by the in vitro kinase assay using kinase-specific antibodies for p38, JNK, and ERK2. The change in expression of hypertrophy marker genes was examined by Northern blot analysis. In VO hypertrophy, the activity of p38 was markedly increased (10-fold), without changing the activity of ERK and JNK. However, during PO hypertrophy, the activity of JNK was significantly increased (two- to sixfold) and depended on the severity of the load. The activity of p38 was not changed in MPO hypertrophy, whereas it was slightly elevated (50%) in hearts with SPO. Similarly, ERK activity was not changed in hearts with MPO, but a transient rise in activity was observed in hearts with SPO. The expression of ANF and beta-MHC genes was elevated in both PO and VO hypertrophy; however, this change was much greater in hearts subjected to PO than VO hypertrophy. Alpha

  19. ARG: Virtual Tool to Teaching Argumentation Theory

    OpenAIRE

    Silva, Nailton; Terrematte, Patrick; Moura, José

    2015-01-01

    Researchers look for new virtual instruments that can improve and maximize traditional forms of teaching and learning. In this paper, we present the ARG system, a virtual tool developed to help the teaching/learning process in argumentation theory, especially in the field of Law. ARG was developed based on Araucaria by Reed and Rowe, Room 5 by Ronald P. Loui, as well on systems such as Argue!-System and ArguMed by Bart Verheij. ARG is a platform for online collaboration and applies the theory...

  20. Focal adhesion kinase regulation in stem cell alignment and spreading on nanofibers.

    Science.gov (United States)

    Andalib, Mohammad Nahid; Lee, Jeong Soon; Ha, Ligyeom; Dzenis, Yuris; Lim, Jung Yul

    2016-05-13

    While electrospun nanofibers have demonstrated the potential for novel tissue engineering scaffolds, very little is known about the molecular mechanism of how cells sense and adapt to nanofibers. Here, we revealed the role of focal adhesion kinase (FAK), one of the key molecular sensors in the focal adhesion complex, in regulating mesenchymal stem cell (MSC) shaping on nanofibers. We produced uniaxially aligned and randomly distributed nanofibers from poly(l-lactic acid) to have the same diameters (about 130 nm) and evaluated MSC behavior on these nanofibers comparing with that on flat PLLA control. C3H10T1/2 murine MSCs exhibited upregulations in FAK expression and phosphorylation (pY397) on nanofibrous cultures as assessed by immunoblotting, and this trend was even greater on aligned nanofibers. MSCs showed significantly elongated and well-spread morphologies on aligned and random nanofibers, respectively. In the presence of FAK silencing via small hairpin RNA (shRNA), cell elongation length in the aligned nanofiber direction (cell major axis length) was significantly decreased, while cells still showed preferred orientation along the aligned nanofibers. On random nanofibers, MSCs with FAK-shRNA showed impaired cell spreading resulting in smaller cell area and higher circularity. Our study provides new data on how MSCs shape their morphologies on aligned and random nanofibrous cultures potentially via FAK-mediated mechanism. PMID:27040763

  1. p53 negatively regulates transcription of the pyruvate dehydrogenase kinase Pdk2.

    Science.gov (United States)

    Contractor, Tanupriya; Harris, Chris R

    2012-01-15

    In cancer cells, the aberrant conversion of pyruvate into lactate instead of acetyl-CoA in the presence of oxygen is known as the Warburg effect. The consequences and mechanisms of this metabolic peculiarity are incompletely understood. Here we report that p53 status is a key determinant of the Warburg effect. Wild-type p53 expression decreased levels of pyruvate dehydrogenase kinase-2 (Pdk2) and the product of its activity, the inactive form of the pyruvate dehydrogenase complex (P-Pdc), both of which are key regulators of pyruvate metabolism. Decreased levels of Pdk2 and P-Pdc in turn promoted conversion of pyruvate into acetyl-CoA instead of lactate. Thus, wild-type p53 limited lactate production in cancer cells unless Pdk2 could be elevated. Together, our results established that wild-type p53 prevents manifestation of the Warburg effect by controlling Pdk2. These findings elucidate a new mechanism by which p53 suppresses tumorigenesis acting at the level of cancer cell metabolism. PMID:22123926

  2. Regulation of Thrombin-Induced Lung Endothelial Cell Barrier Disruption by Protein Kinase C Delta

    Science.gov (United States)

    Xie, Lishi; Chiang, Eddie T.; Kelly, Gabriel T.; Kanteti, Prasad; Singleton, Patrick A.; Camp, Sara M.; Zhou, Tingting; Dudek, Steven M.; Natarajan, Viswanathan; Wang, Ting; Black, Steven M.; Garcia, Joe G. N.; Jacobson, Jeffrey R.

    2016-01-01

    Protein Kinase C (PKC) plays a significant role in thrombin-induced loss of endothelial cell (EC) barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue–specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin), dominant negative PKCδ construct and PKCδ silencing (siRNA). In addition, we identified PKCδ as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ) and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis. PMID:27442243

  3. Regulation of Thrombin-Induced Lung Endothelial Cell Barrier Disruption by Protein Kinase C Delta.

    Directory of Open Access Journals (Sweden)

    Lishi Xie

    Full Text Available Protein Kinase C (PKC plays a significant role in thrombin-induced loss of endothelial cell (EC barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue-specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin, dominant negative PKCδ construct and PKCδ silencing (siRNA. In addition, we identified PKCδ as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis.

  4. SHP1 tyrosine phosphatase negatively regulates NPM-ALK tyrosine kinase signaling.

    Science.gov (United States)

    Honorat, Jean-François; Ragab, Ashraf; Lamant, Laurence; Delsol, Georges; Ragab-Thomas, Jeannie

    2006-05-15

    Anaplastic large-cell lymphoma (ALCL) is frequently associated with the 2;5 translocation and expresses the NPM-ALK fusion protein, which possesses a constitutive tyrosine kinase activity. We analyzed SHP1 tyrosine phosphatase expression and activity in 3 ALK-positive ALCL cell lines (Karpas 299, Cost, and SU-DHL1) and in lymph node biopsies (n = 40). We found an inverse correlation between the level of NPM-ALK phosphorylation and SHP1 phosphatase activity. Pull-down and coimmunoprecipitation experiments demonstrated a SHP1/NPM-ALK association. Furthermore, confocal microscopy performed on ALCL cell lines and biopsy specimens showed the colocalization of the 2 proteins in cytoplasmic bodies containing Y664-phosphorylated NPM-ALK. Dephosphorylation of NPM-ALK by SHP1 demonstrated that NPM-ALK was a SHP1 substrate. Downregulation of SHP1 expression by RNAi in Karpas cells led to hyperphosphorylation of NPM-ALK, STAT3 activation, and increase in cell proliferation. Furthermore, SHP1 overexpression in 3T3 fibroblasts stably expressing NPM-ALK led to the decrease of NPM-ALK phosphorylation, lower cell proliferation, and tumor progression in nude mice. These findings show that SHP1 is a negative regulator of NPM-ALK signaling. The use of tissue microarrays revealed that 50% of ALK-positive ALCLs were positive for SHP1. Our results suggest that SHP1 could be a critical enzyme in ALCL biology and a potential therapeutic target.

  5. Regulation of WRKY46 transcription factor function by mitogen-activated protein kinases in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Arsheed Hussain Sheikh

    2016-02-01

    Full Text Available AbstractMitogen-activated protein kinase (MAPK cascades are central signalling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs, such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defence as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defence.

  6. Transcriptional regulation of oncogenic protein kinase Cϵ (PKCϵ) by STAT1 and Sp1 proteins.

    Science.gov (United States)

    Wang, HongBin; Gutierrez-Uzquiza, Alvaro; Garg, Rachana; Barrio-Real, Laura; Abera, Mahlet B; Lopez-Haber, Cynthia; Rosemblit, Cinthia; Lu, Huaisheng; Abba, Martin; Kazanietz, Marcelo G

    2014-07-11

    Overexpression of PKCϵ, a kinase associated with tumor aggressiveness and widely implicated in malignant transformation and metastasis, is a hallmark of multiple cancers, including mammary, prostate, and lung cancer. To characterize the mechanisms that control PKCϵ expression and its up-regulation in cancer, we cloned an ∼ 1.6-kb promoter segment of the human PKCϵ gene (PRKCE) that displays elevated transcriptional activity in cancer cells. A comprehensive deletional analysis established two regions rich in Sp1 and STAT1 sites located between -777 and -105 bp (region A) and -921 and -796 bp (region B), respectively, as responsible for the high transcriptional activity observed in cancer cells. A more detailed mutagenesis analysis followed by EMSA and ChIP identified Sp1 sites in positions -668/-659 and -269/-247 as well as STAT1 sites in positions -880/-869 and -793/-782 as the elements responsible for elevated promoter activity in breast cancer cells relative to normal mammary epithelial cells. RNAi silencing of Sp1 and STAT1 in breast cancer cells reduced PKCϵ mRNA and protein expression, as well as PRKCE promoter activity. Moreover, a strong correlation was found between PKCϵ and phospho-Ser-727 (active) STAT1 levels in breast cancer cells. Our results may have significant implications for the development of approaches to target PKCϵ and its effectors in cancer therapeutics.

  7. Drosophila Spidey/Kar Regulates Oenocyte Growth via PI3-Kinase Signaling.

    Science.gov (United States)

    Cinnamon, Einat; Makki, Rami; Sawala, Annick; Wickenberg, Leah P; Blomquist, Gary J; Tittiger, Claus; Paroush, Ze'ev; Gould, Alex P

    2016-08-01

    Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. Here, we focus on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. We now show that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, we show that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, the findings in this study show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes. PMID:27500738

  8. Kinase programs spatiotemporally regulate gap junction assembly and disassembly: Effects on wound repair.

    Science.gov (United States)

    Solan, Joell L; Lampe, Paul D

    2016-02-01

    Gap junctions are highly ordered plasma membrane domains that are constantly assembled, remodeled and turned over due to the short half-life of connexins, the integral membrane proteins that form gap junctions. Connexin 43 (Cx43), by far the most widely expressed connexin, is phosphorylated at multiple serine residues in the cytoplasmic, C-terminal region allowing for exquisite cellular control over gap junctional communication. This is evident during epidermal wounding where spatiotemporal changes in connexin expression occur as cells are instructed whether to die, proliferate or migrate to promote repair. Early gap junctional communication is required for initiation of keratinocyte migration, but accelerated Cx43 turnover is also critical for proper wound healing at later stages. These events are controlled via a "kinase program" where sequential phosphorylation of Cx43 leads to reductions in Cx43's half-life and significant depletion of gap junctions from the plasma membrane within several hours. The complex regulation of gap junction assembly and turnover affords several steps where intervention might speed wound healing. PMID:26706150

  9. Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana.

    Science.gov (United States)

    Sheikh, Arsheed H; Eschen-Lippold, Lennart; Pecher, Pascal; Hoehenwarter, Wolfgang; Sinha, Alok K; Scheel, Dierk; Lee, Justin

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense. PMID:26870073

  10. Molecular mechanism and evolution of guanylate kinase regulation by (p)ppGpp.

    Science.gov (United States)

    Liu, Kuanqing; Myers, Angela R; Pisithkul, Tippapha; Claas, Kathy R; Satyshur, Kenneth A; Amador-Noguez, Daniel; Keck, James L; Wang, Jue D

    2015-02-19

    The nucleotide (p)ppGpp mediates bacterial stress responses, but its targets and underlying mechanisms of action vary among bacterial species and remain incompletely understood. Here, we characterize the molecular interaction between (p)ppGpp and guanylate kinase (GMK), revealing the importance of this interaction in adaptation to starvation. Combining structural and kinetic analyses, we show that (p)ppGpp binds the GMK active site and competitively inhibits the enzyme. The (p)ppGpp-GMK interaction prevents the conversion of GMP to GDP, resulting in GMP accumulation upon amino acid downshift. Abolishing this interaction leads to excess (p)ppGpp and defective adaptation to amino acid starvation. A survey of GMKs from phylogenetically diverse bacteria shows that the (p)ppGpp-GMK interaction is conserved in members of Firmicutes, Actinobacteria, and Deinococcus-Thermus, but not in Proteobacteria, where (p)ppGpp regulates RNA polymerase (RNAP). We propose that GMK is an ancestral (p)ppGpp target and RNAP evolved more recently as a direct target in Proteobacteria.

  11. AMP kinase regulates ligand-gated K-ATP channels in substantia nigra dopamine neurons.

    Science.gov (United States)

    Shen, Ke-Zhong; Wu, Yan-Na; Munhall, Adam C; Johnson, Steven W

    2016-08-25

    AMP-activated protein kinase (AMPK) is a master enzyme that regulates ATP-sensitive K(+) (K-ATP) channels in pancreatic beta-cells and cardiac myocytes. We used patch pipettes to record currents and potentials to investigate effects of AMPK on K-ATP currents in substantia nigra compacta (SNC) dopamine neurons in slices of rat midbrain. When slices were superfused repeatedly with the K-ATP channel opener diazoxide, we were surprised to find that diazoxide currents gradually increased in magnitude, reaching 300% of the control value 60min after starting whole-cell recording. However, diazoxide current increased significantly more, to 472% of control, when recorded in the presence of the AMPK activator A769662. Moreover, superfusing the slice with the AMPK blocking agent dorsomorphin significantly reduced diazoxide current to 38% of control. Control experiments showed that outward currents evoked by the K-ATP channel opener NN-414 also increased over time, but not currents evoked by the GABAB agonist baclofen. Delaying the application of diazoxide after starting whole-cell recording correlated with augmentation of current. Loose-patch recording showed that diazoxide produced a 34% slowing of spontaneous firing rate that did not intensify with repeated applications of diazoxide. However, superfusion with A769662 significantly augmented the inhibitory effect of diazoxide on firing rate. We conclude that K-ATP channel function is augmented by AMPK, which is activated during the process of making whole-cell recordings. Our results suggest that AMPK and K-ATP interactions may play an important role in regulating dopamine neuronal excitability. PMID:27267246

  12. AMP kinase regulates ligand-gated K-ATP channels in substantia nigra dopamine neurons.

    Science.gov (United States)

    Shen, Ke-Zhong; Wu, Yan-Na; Munhall, Adam C; Johnson, Steven W

    2016-08-25

    AMP-activated protein kinase (AMPK) is a master enzyme that regulates ATP-sensitive K(+) (K-ATP) channels in pancreatic beta-cells and cardiac myocytes. We used patch pipettes to record currents and potentials to investigate effects of AMPK on K-ATP currents in substantia nigra compacta (SNC) dopamine neurons in slices of rat midbrain. When slices were superfused repeatedly with the K-ATP channel opener diazoxide, we were surprised to find that diazoxide currents gradually increased in magnitude, reaching 300% of the control value 60min after starting whole-cell recording. However, diazoxide current increased significantly more, to 472% of control, when recorded in the presence of the AMPK activator A769662. Moreover, superfusing the slice with the AMPK blocking agent dorsomorphin significantly reduced diazoxide current to 38% of control. Control experiments showed that outward currents evoked by the K-ATP channel opener NN-414 also increased over time, but not currents evoked by the GABAB agonist baclofen. Delaying the application of diazoxide after starting whole-cell recording correlated with augmentation of current. Loose-patch recording showed that diazoxide produced a 34% slowing of spontaneous firing rate that did not intensify with repeated applications of diazoxide. However, superfusion with A769662 significantly augmented the inhibitory effect of diazoxide on firing rate. We conclude that K-ATP channel function is augmented by AMPK, which is activated during the process of making whole-cell recordings. Our results suggest that AMPK and K-ATP interactions may play an important role in regulating dopamine neuronal excitability.

  13. Regulation of the S-locus receptor kinase and self-incompatibility in Arabidopsis thaliana.

    Science.gov (United States)

    Strickler, Susan R; Tantikanjana, Titima; Nasrallah, June B

    2013-02-01

    Intraspecific mate selectivity often is enforced by self-incompatibility (SI), a barrier to self-pollination that inhibits productive pollen-pistil interactions. In the Brassicaceae, SI specificity is determined by two highly-polymorphic proteins: the stigmatic S-locus receptor kinase (SRK) and its pollen coat-localized ligand, the S-locus cysteine-rich protein (SCR). Arabidopsis thaliana is self fertile, but several of its accessions can be made to express SI, albeit to various degrees, by transformation with functional SRK-SCR gene pairs isolated from its close self-incompatible relative, Arabidopsis lyrata. Here, we use a newly identified induced mutation that suppresses the SI phenotype in stigmas of SRK-SCR transformants of the Col-0 accession to investigate the regulation of SI and the SRK transgene. This mutation disrupts NRPD1a, a gene that encodes a plant-specific nuclear RNA polymerase required for genomic methylation and production of some types of silencing RNAs. We show that NRPD1a, along with the RNA-dependent RNA polymerase RDR2, is required for SI in some A. thaliana accessions. We also show that Col-0 nrpd1a mutants exhibit decreased accumulation of SRK transcripts in stigmas, which is not, however, responsible for loss of SI in these plants. Together, our analysis of the nrpd1a mutation and of SRK promoter activity in various accessions reveals that the SRK transgene is subject to several levels of regulation, which vary substantially by tissue type and by accession. This study thus helps explain the well-documented differences in expression of SI exhibited by SRK-SCR transformants of different A. thaliana accessions.

  14. Sonic hedgehog regulates osteoblast function by focal adhesion kinase signaling in the process of fracture healing.

    Directory of Open Access Journals (Sweden)

    Yuu Horikiri

    Full Text Available Several biological studies have indicated that hedgehog signaling plays an important role in osteoblast proliferation and differentiation, and sonic hedgehog (SHH expression is positively correlated with phosphorylated focal adhesion kinase (FAK Tyr(397. However, the relationship between them and their role in the process of normal fracture repair has not been clarified yet. Immunohistochemical analysis revealed that SHH and pFAK Tyr(397 were expressed in bone marrow cells and that pFAK Tyr(397 was also detected in ALP-positive osteoblasts near the TRAP-positive osteoclasts in the fracture site in the ribs of mice on day 5 after fracture. SHH and pFAK Tyr(397 were detectable in osteoblasts near the hypertrophic chondrocytes on day 14. In vitro analysis showed that SHH up-regulated the expression of FAK mRNA and pFAK Tyr(397 time dependently in osteoblastic MC3T3-E1 cells. Functional analysis revealed that 5 lentivirus encoding short hairpin FAK RNAs (shFAK-infected MC3T3-E1 cell groups displayed a round morphology and decreased proliferation, adhesion, migration, and differentiation. SHH stimulated the proliferation and differentiation of MC3T3-E1 cells, but had no effect on the shFAK-infected cells. SHH also stimulated osteoclast formation in a co-culture system containing MC3T3-E1 and murine CD11b(+ bone marrow cells, but did not affect the shFAK-infected MC3T3-E1 co-culture group. These data suggest that SHH signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture and regulated their proliferation and differentiation, as well as osteoclast formation, via FAK signaling.

  15. TRESK background K(+ channel is inhibited by PAR-1/MARK microtubule affinity-regulating kinases in Xenopus oocytes.

    Directory of Open Access Journals (Sweden)

    Gabriella Braun

    Full Text Available TRESK (TWIK-related spinal cord K(+ channel, KCNK18 is a major background K(+ channel of sensory neurons. Dominant-negative mutation of TRESK is linked to familial migraine. This important two-pore domain K(+ channel is uniquely activated by calcineurin. The calcium/calmodulin-dependent protein phosphatase directly binds to the channel and activates TRESK current several-fold in Xenopus oocytes and HEK293 cells. We have recently shown that the kinase, which is responsible for the basal inhibition of the K(+ current, is sensitive to the adaptor protein 14-3-3. Therefore we have examined the effect of the 14-3-3-inhibited PAR-1/MARK, microtubule-associated-protein/microtubule affinity-regulating kinase on TRESK in the Xenopus oocyte expression system. MARK1, MARK2 and MARK3 accelerated the return of TRESK current to the resting state after the calcium-dependent activation. Several other serine-threonine kinase types, generally involved in the modulation of other ion channels, failed to influence TRESK current recovery. MARK2 phosphorylated the primary determinant of regulation, the cluster of three adjacent serine residues (S274, 276 and 279 in the intracellular loop of mouse TRESK. In contrast, serine 264, the 14-3-3-binding site of TRESK, was not phosphorylated by the kinase. Thus MARK2 selectively inhibits TRESK activity via the S274/276/279 cluster, but does not affect the direct recruitment of 14-3-3 to the channel. TRESK is the first example of an ion channel phosphorylated by the dynamically membrane-localized MARK kinases, also known as general determinants of cellular polarity. These results raise the possibility that microtubule dynamics is coupled to the regulation of excitability in the neurons, which express TRESK background potassium channel.

  16. Glycogen Synthase Kinase-3 regulates IGFBP-1 gene transcription through the Thymine-rich Insulin Response Element

    Directory of Open Access Journals (Sweden)

    Marquez Rodolfo

    2004-09-01

    Full Text Available Abstract Background Hepatic expression of several gene products involved in glucose metabolism, including phosphoenolpyruvate carboxykinase (PEPCK, glucose-6-phosphatase (G6Pase and insulin-like growth factor binding protein-1 (IGFBP-1, is rapidly and completely inhibited by insulin. This inhibition is mediated through the regulation of a DNA element present in each of these gene promoters, that we call the Thymine-rich Insulin Response Element (TIRE. The insulin signalling pathway that results in the inhibition of these gene promoters requires the activation of phosphatidylinositol 3-kinase (PI 3-kinase. However, the molecules that connect PI 3-kinase to these gene promoters are not yet fully defined. Glycogen Synthase Kinase 3 (GSK-3 is inhibited following activation of PI 3-kinase. We have shown previously that inhibitors of GSK-3 reduce the activity of two TIRE-containing gene promoters (PEPCK and G6Pase, whose products are required for gluconeogenesis. Results In this report we demonstrate that in H4IIE-C3 cells, four distinct classes of GSK-3 inhibitor mimic the effect of insulin on a third TIRE-containing gene, IGFBP-1. We identify the TIRE as the minimum requirement for inhibition by these agents, and demonstrate that the target of GSK-3 is unlikely to be the postulated TIRE-binding protein FOXO-1. Importantly, overexpression of GSK-3 in cells reduces the insulin regulation of TIRE activity as well as endogenous IGFBP-1 expression. Conclusions These results implicate GSK-3 as an intermediate in the pathway from the insulin receptor to the TIRE. Indeed, this is the first demonstration of an absolute requirement for GSK-3 inhibition in insulin regulation of gene transcription. These data support the potential use of GSK-3 inhibitors in the treatment of insulin resistant states such as Type 2 diabetes mellitus, but suggest that it will be important to identify all TIRE-containing genes to assess potential side effects of these agents.

  17. Regulation of glycogen synthase kinase-3{beta} (GSK-3{beta}) after ionizing radiation; Regulation der Glykogen Synthase Kinase-3{beta} (GSK-3{beta}) nach ionisierender Strahlung

    Energy Technology Data Exchange (ETDEWEB)

    Boehme, K.A.

    2006-12-15

    Glycogen Synthase Kinase-3{beta} (GSK-3{beta}) phosphorylates the Mdm2 protein in the central domain. This phosphorylation is absolutely required for p53 degradation. Ionizing radiation inactivates GSK-3{beta} by phosphorylation at serine 9 and in consequence prevents Mdm2 mediated p53 degradation. During the work for my PhD I identified Akt/PKB as the kinase that phosphorylates GSK-3{beta} at serine 9 after ionizing radiation. Ionizing radiation leads to phosphorylation of Akt/PKB at threonine 308 and serine 473. The PI3 Kinase inhibitor LY294002 completely abolished Akt/PKB serine 473 phosphorylation and prevented the induction of GSK-3{beta} serine 9 phosphorylation after ionizing radiation. Interestingly, the most significant activation of Akt/PKB after ionizing radiation occurred in the nucleus while cytoplasmic Akt/PKB was only weakly activated after radiation. By using siRNA, I showed that Akt1/PKBa, but not Akt2/PKB{beta}, is required for phosphorylation of GSK- 3{beta} at serine 9 after ionizing radiation. Phosphorylation and activation of Akt/PKB after ionizing radiation depends on the DNA dependent protein kinase (DNA-PK), a member of the PI3 Kinase family, that is activated by free DNA ends. Both, in cells from SCID mice and after knockdown of the catalytic subunit of DNA-PK by siRNA in osteosarcoma cells, phosphorylation of Akt/PKB at serine 473 and of GSK-3{beta} at serine 9 was completely abolished. Consistent with the principle that phosphorylation of GSK-3 at serine 9 contributes to p53 stabilization after radiation, the accumulation of p53 in response to ionizing radiation was largely prevented by downregulation of DNA-PK. From these results I conclude, that ionizing radiation induces a signaling cascade that leads to Akt1/PKBa activation mediated by DNA-PK dependent phosphorylation of serine 473. After activation Akt1/PKBa phosphorylates and inhibits GSK-3{beta} in the nucleus. The resulting hypophosphorylated form of Mdm2 protein is no longer

  18. Huntingtin Subcellular Localisation Is Regulated by Kinase Signalling Activity in the StHdhQ111 Model of HD

    Science.gov (United States)

    Bowles, Kathryn R.; Brooks, Simon P.; Dunnett, Stephen B.; Jones, Lesley

    2015-01-01

    Huntington’s disease is a neurodegenerative disorder characterised primarily by motor abnormalities, and is caused by an expanded polyglutamine repeat in the huntingtin protein. Huntingtin dynamically shuttles between subcellular compartments, and the mutant huntingtin protein is mislocalised to cell nuclei, where it may interfere with nuclear functions, such as transcription. However, the mechanism by which mislocalisation of mutant huntingtin occurs is currently unknown. An immortalised embryonic striatal cell model of HD (StHdhQ111) was stimulated with epidermal growth factor in order to determine whether the subcellular localisation of huntingtin is dependent on kinase signalling pathway activation. Aberrant phosphorylation of AKT and MEK signalling pathways was identified in cells carrying mutant huntingtin. Activity within these pathways was found to contribute to the regulation of huntingtin and mutant huntingtin localisation, as well as to the expression of immediate-early genes. We propose that altered kinase signalling is a phenotype of Huntington’s disease that occurs prior to cell death; specifically, that altered kinase signalling may influence huntingtin localisation, which in turn may impact upon nuclear processes such as transcriptional regulation. Aiming to restore the balance of activity between kinase signalling networks may therefore prove to be an effective approach to delaying Huntington’s disease symptom development and progression. PMID:26660732

  19. Balance between Coiled-Coil Stability and Dynamics Regulates Activity of BvgS Sensor Kinase in Bordetella

    Directory of Open Access Journals (Sweden)

    E. Lesne

    2016-03-01

    Full Text Available The two-component system BvgAS controls the expression of the virulence regulon of Bordetella pertussis. BvgS is a prototype of bacterial sensor kinases with extracytoplasmic Venus flytrap perception domains. Following its transmembrane segment, BvgS harbors a cytoplasmic Per-Arnt-Sim (PAS domain and then a predicted 2-helix coiled coil that precede the dimerization-histidine-phosphotransfer domain of the kinase. BvgS homologs have a similar domain organization, or they harbor only a predicted coiled coil between the transmembrane and the dimerization-histidine-phosphotransfer domains. Here, we show that the 2-helix coiled coil of BvgS regulates the enzymatic activity in a mechanical manner. Its marginally stable hydrophobic interface enables a switch between a state of great rotational dynamics in the kinase mode and a more rigid conformation in the phosphatase mode in response to signal perception by the periplasmic domains. We further show that the activity of BvgS is controlled in the same manner if its PAS domain is replaced with the natural α-helical sequences of PAS-less homologs. Clamshell motions of the Venus flytrap domains trigger the shift of the coiled coil’s dynamics. Thus, we have uncovered a general mechanism of regulation for the BvgS family of Venus flytrap-containing two-component sensor kinases.

  20. Serum inducible kinase is a positive regulator of cortical dendrite development and is required for BDNF-promoted dendritic arborization

    Institute of Scientific and Technical Information of China (English)

    Shun-Ling Guo; Guo-He Tan; Shuai Li; Xue-Wen Cheng; Ya Zhou; Yun-Fang Jia; Hui Xiong; Jiong Tao; Zhi-Qi Xiong

    2012-01-01

    Serum inducible kinase (SNK),also known as (p)olo-(l)ike (k)inase 2 (PLK2),is a known regulator of mitosis,synaptogenesis and synaptic homeostasis.However,its role in early cortical development is unknown.Herein,we show that snk is expressed in the cortical plate from embryonic day 14,but not in the ventricular/subventricular zones (VZ/SVZ),and SNK protein localizes to the soma and dendrites of cultured immature cortical neurons.Loss of SNK impaired dendritic but not axonal arborization in a dose-dependent manner and overexpression had opposite effects,both in vitro and in vivo.Overexpression of SNK also caused abnormal branching of the leading process of migrating cortical neurons in electroporated cortices.The kinase activity was necessary for these effects.Extracellular signalregulated kinase (ERK) pathway activity downstream of brain-derived neurotrophic factor (BDNF) stimulation led to increases in SNK protein expression via transcriptional regulation,and this upregulation was necessary for the growth-promoting effect of BDNF on dendritic arborization.Taken together,our results indicate that SNK is essential for dendrite morphogenesis in cortical neurons.

  1. Protein kinase D2 regulates migration and invasion of U87MG glioblastoma cells in vitro

    International Nuclear Information System (INIS)

    Glioblastoma multiforme (GBM) is the most common malignant brain tumor, which, despite combined modality treatment, reoccurs and is invariably fatal for affected patients. Recently, a member of the serine/threonine protein kinase D (PRKD) family, PRKD2, was shown to be a potent mediator of glioblastoma growth. Here we studied the role of PRKD2 in U87MG glioblastoma cell migration and invasion in response to sphingosine-1-phosphate (S1P), an activator of PRKD2 and a GBM mitogen. Time-lapse microscopy demonstrated that random cell migration was significantly diminished in response to PRKD2 silencing. The pharmacological PRKD family inhibitor CRT0066101 decreased chemotactic migration and invasion across uncoated or matrigel-coated Transwell inserts. Silencing of PRKD2 attenuated migration and invasion of U87MG cells even more effectively. In terms of downstream signaling, CRT0066101 prevented PRKD2 autophosphorylation and inhibited p44/42 MAPK and to a smaller extent p54/46 JNK and p38 MAPK activation. PRKD2 silencing impaired activation of p44/42 MAPK and p54/46 JNK, downregulated nuclear c-Jun protein levels and decreased c-JunS73 phosphorylation without affecting the NFκB pathway. Finally, qPCR array analyses revealed that silencing of PRKD2 downregulates mRNA levels of integrin alpha-2 and -4 (ITGA2 and -4), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and matrix metallopeptidase 1 (MMP1). Findings of the present study identify PRKD2 as a potential target to interfere with glioblastoma cell migration and invasion, two major determinants contributing to recurrence of glioblastoma after multimodality treatment. Highlights: • Sphingosine-1-phosphate induces glioma cell migration and invasion. • Part of the effects is mediated by protein kinase D2 (PRKD2) activation. • Inactivation of PRKD2 attenuates glioblastoma cell migration and invasion. • Both, RNAi and pharmacological inhibition of PRKD2 inhibits MAPK

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

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Yuchao; Ramachandran, Anup [Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Breckenridge, David G.; Liles, John T. [Department of Biology, Gilead Sciences, Inc., Foster City, CA (United States); Lebofsky, Margitta [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); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

    2015-07-01

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

  3. Protein signaling and regulation of gene transcription in leukemia: role of the Casein Kinase II-Ikaros axis.

    Science.gov (United States)

    Gowda, Chandrika S; Song, Chunhua; Ding, Yali; Kapadia, Malika; Dovat, Sinisa

    2016-03-01

    Protein signaling and regulation of gene expression are the two major mechanisms that regulate cellular proliferation in leukemia. Discerning the function of these processes is essential for understanding the pathogenesis of leukemia and for developing the targeted therapies. Here, we provide an overview of one of the mechanisms that regulates gene transcription in leukemia. This mechanism involves the direct interaction between Casein Kinase II (CK2) and the Ikaros transcription factor. Ikaros (IKZF1) functions as a master regulator of hematopoiesis and a tumor suppressor in acute lymphoblastic leukemia (ALL). Impaired Ikaros function results in the development of high-risk leukemia. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. In vivo, Ikaros is a target for CK2, a pro-oncogenic kinase. CK2 directly phosphorylates Ikaros at multiple amino acids. Functional experiments showed that CK2-mediated phosphorylation of Ikaros, regulates Ikaros' DNA binding affinity, subcellular localization and protein stability. Recent studies revealed that phosphorylation of Ikaros by CK2 regulates Ikaros binding and repression of the terminal deoxytransferase (TdT) gene in normal thymocytes and in T-cell ALL. Available data suggest that the oncogenic activity of CK2 in leukemia involves functional inactivation of Ikaros and provide a rationale for CK2 inhibitors as a potential treatment for ALL. PMID:26912004

  4. Role of extracellular signal-regulated kinase in regulating expression of interleukin 13 in lymphocytes from an asthmatic rat model

    Institute of Scientific and Technical Information of China (English)

    LI Yuan-yuan; LIU Xian-sheng; LIU Chang; XU Yong-jian; XIONG Wei-xing

    2010-01-01

    Background The extracellular signal-regulated kinase (ERK) is widely expressed in mammal cells and involved in airway proliferation and remodeling in asthma. In this study, we intend to explore the role of ERK in the expression of the Th2 cytokine, interleukin 13 (IL-13) in lymphocytes in asthma.Methods Forty Sprague-Dawley rats were randomly divided into two groups: normal control and asthmatic groups. Peripheral blood lymphocytes were isolated and purified from the blood of each rat and divided into five groups: control, asthmatic lymphocytes, asthmatic cells stimulated with ERK activator epidermal growth factor (EGF), or with ERK inhibitor PD98059, or with EGF and PD98059 together. The expression of phosphorylated-ERK (p-ERK) was observed by immunocvtochemical staining, the expression of ERK mRNA was determined by reverse transcriptase-PCR, IL-13 protein in supernatants was measured by ELISA.Results (1) The ERK mRNA level and the percentage of cells with p-ERK in lymphocytes from asthmatic rats were significantly higher than those in normal controls, and were significantly increased by EGF administration. This effect of EGF was significantly inhibited by PD98059 pretreatment. (2) IL-13 protein in supematants of asthmatic lymphocytes was higher than that produced by normal control lymphocytes, and was significantly increased by EGF treatment. This EGF effect was partly blocked by PD98059 pretreatment. (3) There was a significant positive correlation between the percentage of cells with p-ERK in peripheral blood lymphocytes and IL-13 protein in supematants of lymphocytes from asthmatic rats.Conclusions In asthma the ERK expression and activation levels were increased, as was the protein level of IL-13. The ERK signaling pathway may be involved in the increased expression of the Th2 cytokine IL-13 in asthma.

  5. Regulation of protein kinase Cδ downregulation by protein kinase Cε and mammalian target of rapamycin complex 2

    OpenAIRE

    Basu, Alakananda; Sridharan, Savitha; Persaud, Shalini

    2009-01-01

    Phosphorylation and dephosphorylation of PKCs can regulate their activity, stability and function. We have previously shown that downregulation of PKCδ by tumor promoting phorbol esters was compromised when HeLa cells acquired resistance to cisplatin (HeLa/CP). In the present study, we have used these cells to understand the mechanism of PKCδ downregulation. A brief treatment of HeLa cells with phorbol 12,13-dibutyrate (PDBu) induced phosphorylation of PKCδ at the activation loop (Thr505), tu...

  6. Light-regulated root gravitropism: a role for, and characterization of, a calcium/calmodulin-dependent protein kinase homolog

    Science.gov (United States)

    Lu, Y. T.; Feldman, L. J.

    1997-01-01

    Roots of many species grow downward (orthogravitropism) only when illuminated. Previous work suggests that this is a calcium-regulated response and that both calmodulin and calcium/calmodulin-dependent kinases participate in transducing gravity and light stimuli. A genomic sequence has been obtained for a calcium/calmodulin-dependent kinase homolog (MCK1) expressed in root caps, the site of perception for both light and gravity. This homolog consists of 7265 base pairs and contains 11 exons and 10 introns. Since MCK1 is expressed constitutively in both light and dark, it is unlikely that the light directly affects MCK1 expression, though the activity of the protein may be affected by light. In cultivars showing light-regulated gravitropism, we hypothesize that MCK1, or a homolog, functions in establishing the auxin asymmetry necessary for orthogravitropism.

  7. Phosphorylation of Yeast Pah1 Phosphatidate Phosphatase by Casein Kinase II Regulates Its Function in Lipid Metabolism.

    Science.gov (United States)

    Hsieh, Lu-Sheng; Su, Wen-Min; Han, Gil-Soo; Carman, George M

    2016-05-01

    Pah1 phosphatidate phosphatase in Saccharomyces cerevisiae catalyzes the penultimate step in the synthesis of triacylglycerol (i.e. the production of diacylglycerol by dephosphorylation of phosphatidate). The enzyme playing a major role in lipid metabolism is subject to phosphorylation (e.g. by Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and C) and dephosphorylation (e.g. by Nem1-Spo7) that regulate its cellular location, catalytic activity, and stability/degradation. In this work, we show that Pah1 is a substrate for casein kinase II (CKII); its phosphorylation was time- and dose-dependent and was dependent on the concentrations of Pah1 (Km = 0.23 μm) and ATP (Km = 5.5 μm). By mass spectrometry, truncation analysis, site-directed mutagenesis, phosphopeptide mapping, and phosphoamino acid analysis, we identified that >90% of its phosphorylation occurs on Thr-170, Ser-250, Ser-313, Ser-705, Ser-814, and Ser-818. The CKII-phosphorylated Pah1 was a substrate for the Nem1-Spo7 protein phosphatase and was degraded by the 20S proteasome. The prephosphorylation of Pah1 by protein kinase A or protein kinase C reduced its subsequent phosphorylation by CKII. The prephosphorylation of Pah1 by CKII reduced its subsequent phosphorylation by protein kinase A but not by protein kinase C. The expression of Pah1 with combined mutations of S705D and 7A, which mimic its phosphorylation by CKII and lack of phosphorylation by Pho85-Pho80, caused an increase in triacylglycerol content and lipid droplet number in cells expressing the Nem1-Spo7 phosphatase complex. PMID:27044741

  8. Study of the regulation of Fab1p, a phosphatidylinositol 3-phosphate 5 kinase in Saccharomyces cerevisiae.

    OpenAIRE

    Phelan, J. P.

    2005-01-01

    The Saccharomyces cerevisiae protein Fab1p is the archetypal type III phosphatidyl inositol phosphate kinase. This family of enzymes is universal to all eukaryotes and is responsible for the synthesis of phosphatidylinositol 3,5-bisphosphate from phosphatidylinositol 3- phosphate. In S. cerevisiae , Fab1p regulates a number of cellular processes via the production of phosphatidylinositol 3,5-bisphosphate including: vacuole acidification, protein trafficking to the vacuole lumen, vacuole membr...

  9. Aspergillus nidulans Natural Product Biosynthesis Is Regulated by MpkB, a Putative Pheromone Response Mitogen-Activated Protein Kinase

    International Nuclear Information System (INIS)

    The Aspergillus nidulans putative mitogen-activated protein kinase encoded by mpkB has a role in natural product biosynthesis. An mpkB mutant exhibited a decrease in sterigmatocystin gene expression and low mycotoxin levels. The mutation also affected the expression of genes involved in penicillin and terrequinone A synthesis. mpkB was necessary for normal expression of laeA, which has been found to regulate secondary metabolism gene clusters. (author)

  10. Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels.

    Science.gov (United States)

    Lee, Jaekwang; Han, Young-Eun; Favorov, Oleg; Tommerdahl, Mark; Whitsel, Barry; Lee, C Justin

    2016-04-01

    Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC. PMID:27122993

  11. Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels

    Science.gov (United States)

    Lee, Jaekwang; Han, Young-Eun; Favorov, Oleg; Tommerdahl, Mark; Whitsel, Barry

    2016-01-01

    Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC. PMID:27122993

  12. Reciprocal regulation as a source of ultrasensitivity in two-component systems with a bifunctional sensor kinase.

    Directory of Open Access Journals (Sweden)

    Ronny Straube

    2014-05-01

    Full Text Available Two-component signal transduction systems, where the phosphorylation state of a regulator protein is modulated by a sensor kinase, are common in bacteria and other microbes. In many of these systems, the sensor kinase is bifunctional catalyzing both, the phosphorylation and the dephosphorylation of the regulator protein in response to input signals. Previous studies have shown that systems with a bifunctional enzyme can adjust the phosphorylation level of the regulator protein independently of the total protein concentrations--a property known as concentration robustness. Here, I argue that two-component systems with a bifunctional enzyme may also exhibit ultrasensitivity if the input signal reciprocally affects multiple activities of the sensor kinase. To this end, I consider the case where an allosteric effector inhibits autophosphorylation and, concomitantly, activates the enzyme's phosphatase activity, as observed experimentally in the PhoQ/PhoP and NRII/NRI systems. A theoretical analysis reveals two operating regimes under steady state conditions depending on the effector affinity: If the affinity is low the system produces a graded response with respect to input signals and exhibits stimulus-dependent concentration robustness--consistent with previous experiments. In contrast, a high-affinity effector may generate ultrasensitivity by a similar mechanism as phosphorylation-dephosphorylation cycles with distinct converter enzymes. The occurrence of ultrasensitivity requires saturation of the sensor kinase's phosphatase activity, but is restricted to low effector concentrations, which suggests that this mode of operation might be employed for the detection and amplification of low abundant input signals. Interestingly, the same mechanism also applies to covalent modification cycles with a bifunctional converter enzyme, which suggests that reciprocal regulation, as a mechanism to generate ultrasensitivity, is not restricted to two

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

    Institute of Scientific and Technical Information of China (English)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Kyle K. Biggar

    2015-04-01

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

  15. Intracellular Distribution of Differentially Phosphorylated Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A)

    OpenAIRE

    Kaczmarski, Wojciech; Barua, Madhabi; Mazur-Kolecka, Bozena; Frackowiak, Janusz; Dowjat, Wieslaw; Mehta, Pankaj; Bolton, David; Hwang, Yu-Wen; Rabe, Ausma; Albertini, Giorgio; Wegiel, Jerzy

    2013-01-01

    The gene encoding dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is located within the Down syndrome (DS) critical region of chromosome 21. DYRK1A interacts with a plethora of substrates in the cytosol, cytoskeleton, and nucleus. Its overexpression is a contributing factor to the developmental alterations and age-associated pathology observed in DS. We hypothesized that the intracellular distribution of DYRK1A and cell-compartment–specific functions a...

  16. Regulation of the Tyrosine Kinase Pyk2 by Calcium Is through Production of Reactive Oxygen Species in Cytotoxic T Lymphocytes*

    OpenAIRE

    Lysechko, Tara L.; Cheung, Samuel M. S.; Ostergaard, Hanne L.

    2010-01-01

    Pyk2 was identified as a Ca2+-dependent kinase, however, the regulation of Pyk2 by Ca2+ in T cells remains controversial. We found that Ca2+ mobilization preferentially induced Pyk2 phosphorylation in cytotoxic T lymphocytes (CTL). Furthermore, Pyk2 phosphorylation in CTL was not absolutely Ca2+ dependent but relied on the strength of T cell receptor stimulation. Ionomycin-stimulated Pyk2 phosphorylation did not require calmodulin activity, because phosphorylation was not inhibited by the cal...

  17. 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 ...

  18. A novel functional link between MAP kinase cascades and the Ras/cAMP pathway that regulates survival.

    Science.gov (United States)

    Cherkasova, Vera A; McCully, Ryan; Wang, Yunmei; Hinnebusch, Alan; Elion, Elaine A

    2003-07-15

    In mammalian cells, Ras regulates multiple effectors, including activators of mitogen-activated protein kinase (MAPK) cascades, phosphatidylinositol-3-kinase, and guanine nucleotide exchange factors (GEFs) for RalGTPases. In S. cerevisiae, Ras regulates the Kss1 MAPK cascade that promotes filamentous growth and cell integrity, but its major function is to activate adenylyl cyclase and control proliferation and survival ([; see Figure S1 in the Supplemental Data available with this article online). Previous work hints that the mating Fus3/Kss1 MAPK cascade cross-regulates the Ras/cAMP pathway during growth and mating, but direct evidence is lacking. Here, we report that Kss1 and Fus3 act upstream of the Ras/cAMP pathway to regulate survival. Loss of Fus3 increases cAMP and causes poor long-term survival and resistance to stress. These effects are dependent on Kss1 and Ras2. Activation of Kss1 by a hyperactive Ste11 MAPKKK also increases cAMP, but mating receptor/scaffold activation has little effect and may therefore insulate the MAPKs from cross-regulation. Catalytically inactive Fus3 represses cAMP by blocking accumulation of active Kss1 and by another function also shared by Kss1. The conserved RasGEF Cdc25 is a likely control point, because Kss1 and Fus3 complexes associate with and phosphorylate Cdc25. Cross-regulation of Cdc25 may be a general way that MAPKs control Ras signaling networks. PMID:12867033

  19. TCR-Induced Akt Serine 473 Phosphorylation Is Regulated by Protein Kinase C-Alpha

    OpenAIRE

    Yang, Lifen; Qiao, Guilin; Ying, Haiyan; Zhang, Jian; Yin, Fei

    2010-01-01

    Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser473 in the hydrophobic motif, along with Thr308 in its activation loop, is considered necessary for Akt function. It is widely accepted that Phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr308, but the kinase(s) responsible for phosphorylating Akt at Ser473 (PDK-2) remains elusive. The existence of PDK-2 is considered to be spe...

  20. The Vip1 inositol polyphosphate kinase family regulates polarized growth and modulates the microtubule cytoskeleton in fungi.

    Directory of Open Access Journals (Sweden)

    Jennifer Pöhlmann

    2014-09-01

    Full Text Available Microtubules (MTs are pivotal for numerous eukaryotic processes ranging from cellular morphogenesis, chromosome segregation to intracellular transport. Execution of these tasks requires intricate regulation of MT dynamics. Here, we identify a new regulator of the Schizosaccharomyces pombe MT cytoskeleton: Asp1, a member of the highly conserved Vip1 inositol polyphosphate kinase family. Inositol pyrophosphates generated by Asp1 modulate MT dynamic parameters independent of the central +TIP EB1 and in a dose-dependent and cellular-context-dependent manner. Importantly, our analysis of the in vitro kinase activities of various S. pombe Asp1 variants demonstrated that the C-terminal phosphatase-like domain of the dual domain Vip1 protein negatively affects the inositol pyrophosphate output of the N-terminal kinase domain. These data suggest that the former domain has phosphatase activity. Remarkably, Vip1 regulation of the MT cytoskeleton is a conserved feature, as Vip1-like proteins of the filamentous ascomycete Aspergillus nidulans and the distantly related pathogenic basidiomycete Ustilago maydis also affect the MT cytoskeleton in these organisms. Consistent with the role of interphase MTs in growth zone selection/maintenance, all 3 fungal systems show aspects of aberrant cell morphogenesis. Thus, for the first time we have identified a conserved biological process for inositol pyrophosphates.

  1. Organism-adapted specificity of the allosteric regulation of pyruvate kinase in lactic acid bacteria.

    Directory of Open Access Journals (Sweden)

    Nadine Veith

    Full Text Available Pyruvate kinase (PYK is a critical allosterically regulated enzyme that links glycolysis, the primary energy metabolism, to cellular metabolism. Lactic acid bacteria rely almost exclusively on glycolysis for their energy production under anaerobic conditions, which reinforces the key role of PYK in their metabolism. These organisms are closely related, but have adapted to a huge variety of native environments. They include food-fermenting organisms, important symbionts in the human gut, and antibiotic-resistant pathogens. In contrast to the rather conserved inhibition of PYK by inorganic phosphate, the activation of PYK shows high variability in the type of activating compound between different lactic acid bacteria. System-wide comparative studies of the metabolism of lactic acid bacteria are required to understand the reasons for the diversity of these closely related microorganisms. These require knowledge of the identities of the enzyme modifiers. Here, we predict potential allosteric activators of PYKs from three lactic acid bacteria which are adapted to different native environments. We used protein structure-based molecular modeling and enzyme kinetic modeling to predict and validate potential activators of PYK. Specifically, we compared the electrostatic potential and the binding of phosphate moieties at the allosteric binding sites, and predicted potential allosteric activators by docking. We then made a kinetic model of Lactococcus lactis PYK to relate the activator predictions to the intracellular sugar-phosphate conditions in lactic acid bacteria. This strategy enabled us to predict fructose 1,6-bisphosphate as the sole activator of the Enterococcus faecalis PYK, and to predict that the PYKs from Streptococcus pyogenes and Lactobacillus plantarum show weaker specificity for their allosteric activators, while still having fructose 1,6-bisphosphate play the main activator role in vivo. These differences in the specificity of allosteric

  2. Protein implicated in nonsyndromic mental retardation regulates protein kinase A (PKA) activity

    KAUST Repository

    Al-Tawashi, Azza

    2012-02-28

    Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacitytoformdendritesandsynapsesinculture. Atthebiochemical level,CC2D1Atransduces signals to the cyclic adenosine 3?,5?-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation. 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Class IA phosphatidylinositol 3-kinase p110α regulates phagosome maturation.

    Directory of Open Access Journals (Sweden)

    Emily P Thi

    Full Text Available Of the various phosphatidylinositol 3- kinases (PI3Ks, only the class III enzyme Vps34 has been shown to regulate phagosome maturation. During studies of phagosome maturation in THP-1 cells deficient in class IA PI3K p110α, we discovered that this PI3K isoform is required for vacuole maturation to progress beyond acquisition of Rab7 leading to delivery of lysosomal markers. Bead phagosomes from THP-1 cells acquired p110α and contained PI3P and PI(3,4,5P3; however, p110α and PI(3,4,5P3 levels in phagosomes from p110α knockdown cells were decreased. Phagosomes from p110α knock down cells showed normal acquisition of both Rab5 and EEA-1, but were markedly deficient in the lysosomal markers LAMP-1 and LAMP-2, and the lysosomal hydrolase, β-galactosidase. Phagosomes from p110α deficient cells also displayed impaired fusion with Texas Red dextran-loaded lysosomes. Despite lacking lysosomal components, phagosomes from p110α deficient cells recruited normal levels of Rab7, Rab-interacting lysosomal protein (RILP and homotypic vacuole fusion and protein sorting (HOPs components Vps41 and Vps16. The latter observations demonstrated that phagosomal Rab7 was active and capable of recruiting effectors involved in membrane fusion. Nevertheless, active Rab7 was not sufficient to bring about the delivery of lysosomal proteins to the maturing vacuole, which is shown for the first time to be dependent on a class I PI3K.

  4. Extracellular Microvesicles from Astrocytes Contain Functional Glutamate Transporters: Regulation by Protein Kinase C and Cell Activation

    Directory of Open Access Journals (Sweden)

    Romain-Daniel eGosselin

    2013-12-01

    Full Text Available Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes and because the Protein Kinase C (PKC family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA reorganizes EAAT-1 distribution and reduces functional [3H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [3H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release.

  5. Structural basis and evolution of redox regulation in plant adenosine-5;#8242;-phosphosulfate kinase

    Energy Technology Data Exchange (ETDEWEB)

    Ravilious, Geoffrey E.; Nguyen, Amelia; Francois, Julie A.; Jez, Joseph M. (WU)

    2012-05-08

    Adenosine-5'-phosphosulfate (APS) kinase (APSK) catalyzes the phosphorylation of APS to 3'-phospho-APS (PAPS). In Arabidopsis thaliana, APSK is essential for reproductive viability and competes with APS reductase to partition sulfate between the primary and secondary branches of the sulfur assimilatory pathway; however, the biochemical regulation of APSK is poorly understood. The 1.8-{angstrom} resolution crystal structure of APSR from A. thaliana (AtAPSK) in complex with {beta},{gamma}-imidoadenosine-5'-triphosphate, Mg{sup 2+}, and APS provides a view of the Michaelis complex for this enzyme and reveals the presence of an intersubunit disulfide bond between Cys86 and Cys119. Functional analysis of AtAPSK demonstrates that reduction of Cys86-Cys119 resulted in a 17-fold higher kcat/Km and a 15-fold increase in Ki for substrate inhibition by APS compared with the oxidized enzyme. The C86A/C119A mutant was kinetically similar to the reduced WT enzyme. Gel- and activity-based titrations indicate that the midpoint potential of the disulfide in AtAPSK is comparable to that observed in APS reductase. Both cysteines are invariant among the APSK from plants, but not other organisms, which suggests redox-control as a unique regulatory feature of the plant APSK. Based on structural, functional, and sequence analyses, we propose that the redox-sensitive APSK evolved after bifurcation of the sulfur assimilatory pathway in the green plant lineage and that changes in redox environment resulting from oxidative stresses may affect partitioning of APS into the primary and secondary thiol metabolic routes by having opposing effects on APSK and APS reductase in plants.

  6. Dietary fish oil blocks carcinogen-induced down-regulation of colonic protein kinase C isozymes.

    Science.gov (United States)

    Jiang, Y H; Lupton, J R; Chapkin, R S

    1997-02-01

    In order to elucidate the influence of dietary constituents on colonic intracellular signal transduction, the effect of different fats on rat colonic epithelial protein kinase C (PKC) alpha (classical), delta (novel) and lambda-zeta (atypical) expression was determined in carcinogen-treated animals. Sprague-Dawley rats were provided with one of two fats (corn oil and fish oil); plus or minus the carcinogen azoxymethane (AOM) and killed at two time points (15 and 37 weeks) in a 2x2x2 factorial design. At 5 and 6 weeks of age, animals were injected s.c. with either AOM at a dose of 15 mg/kg body weight or saline once a week for 2 weeks and continued on the same diet until termination of the study. At 15 and 37 weeks after the second injection, 10 rats from each treatment group were killed. Colonic PKC alpha, delta and lambda-zeta steady-state protein and mRNA levels were determined using immunoblotting and relative quantitative polymerase chain reaction, respectively. Colonic mucosa from rats injected with AOM had significantly suppressed membrane and cytosolic PKC alpha and cytosolic lambda-zeta protein levels (P fish oil diets had significantly higher (P protein levels relative to animals fed corn oil diets. However, the effect of diet and AOM on the steady-state expression of PKC alpha, delta and zeta mRNA was not consistent with changes in the respective isozyme protein levels, suggesting regulation at the post-transcriptional level. These data demonstrate that dietary fish oil blocks the carcinogen-induced decrease in the steady-state levels of colonic mucosal PKC delta and lambda-zeta, which may in part explain why this fat source protects against colon cancer development.

  7. Protein kinase C, focal adhesions and the regulation of cell migration

    DEFF Research Database (Denmark)

    Fogh, Betina S; Multhaupt, Hinke A B; Couchman, John Robert

    2014-01-01

    and adhesion turnover. Focal adhesions, or focal contacts, are widespread organelles at the cell-matrix interface. They arise as a result of receptor interactions with matrix ligands, together with clustering. Recent analysis shows that focal adhesions contain a very large number of protein components......Cell adhesion to extracellular matrix is a complex process involving protrusive activity driven by the actin cytoskeleton, engagement of specific receptors, followed by signaling and cytoskeletal organization. Thereafter, contractile and endocytic/recycling activities may facilitate migration...... in their intracellular compartment. Among these are tyrosine kinases, which have received a great deal of attention, whereas the serine/threonine kinase protein kinase C has received much less. Here the status of protein kinase C in focal adhesions and cell migration is reviewed, together with discussion of its roles...

  8. The AMPK-related kinase SNARK regulates muscle mass and myocyte survival

    OpenAIRE

    Lessard, Sarah J.; Rivas, Donato A.; So, Kawai; Koh, Ho-Jin; André Lima de QUEIROZ; Hirshman, Michael F.; Fielding, Roger A.; Goodyear, Laurie J.

    2015-01-01

    The maintenance of skeletal muscle mass is critical for sustaining health; however, the mechanisms responsible for muscle loss with aging and chronic diseases, such as diabetes and obesity, are poorly understood. We found that expression of a member of the AMPK-related kinase family, the SNF1-AMPK-related kinase (SNARK, also known as NUAK2), increased with muscle cell differentiation. SNARK expression increased in skeletal muscles from young mice exposed to metabolic stress and in muscles fro...

  9. Protein kinase C is involved in regulation of Ca2+ channels in plasmalemma of Nitella syncarpa.

    Science.gov (United States)

    Zherelova, O M

    1989-01-01

    Ca2+ current recordings have been made on Nitella syncarpa cells using the intracellular perfusion and the voltage-clamp technique. TPA (12-O-tetradecanoylphorbol-13-acetate), a substance capable of activating protein kinase C from plasmalemma of Nitella cells, modulates voltage-dependent Ca2+ channels. Polymixin B, inhibitor of protein kinase C, blocks the Nitella plasmalemma Ca2+ channels; the rate of channel blockage depends on the concentration and exposure time of the substance. PMID:2536617

  10. Use of a Semisynthetic Epitope to Probe Histidine Kinase Activity and Regulation

    OpenAIRE

    Carlson, Hans K.; Plate, Lars; Price, Mark S.; Allen, Jasmina J.; Shokat, Kevan M.; Marletta, Michael A.

    2009-01-01

    Histidine-aspartic acid phosphotransfer pathways are central components of prokaryotic signal transduction pathways, and are also found in many eukaryotes. Tools to study histidine kinases, however, are currently quite limited. In this paper, we present a new tool to study histidine-aspartic acid phosphotransfer pathways. We show that many histidine kinases will accept ATPγS as a substrate to form a stable thiophosphohistidine, even when they do not form stable phosphohistidines using the nat...

  11. Chapter Three - Ubiquitination and Protein Turnover of G-Protein-Coupled Receptor Kinases in GPCR Signaling and Cellular Regulation.

    Science.gov (United States)

    Penela, P

    2016-01-01

    G-protein-coupled receptors (GPCRs) are responsible for regulating a wide variety of physiological processes, and distinct mechanisms for GPCR inactivation exist to guarantee correct receptor functionality. One of the widely used mechanisms is receptor phosphorylation by specific G-protein-coupled receptor kinases (GRKs), leading to uncoupling from G proteins (desensitization) and receptor internalization. GRKs and β-arrestins also participate in the assembly of receptor-associated multimolecular complexes, thus initiating alternative G-protein-independent signaling events. In addition, the abundant GRK2 kinase has diverse "effector" functions in cellular migration, proliferation, and metabolism homeostasis by means of the phosphorylation or interaction with non-GPCR partners. Altered expression of GRKs (particularly of GRK2 and GRK5) occurs during pathological conditions characterized by impaired GPCR signaling including inflammatory syndromes, cardiovascular disease, and tumor contexts. It is increasingly appreciated that different pathways governing GRK protein stability play a role in the modulation of kinase levels in normal and pathological conditions. Thus, enhanced GRK2 degradation by the proteasome pathway occurs upon GPCR stimulation, what allows cellular adaptation to chronic stimulation in a physiological setting. β-arrestins participate in this process by facilitating GRK2 phosphorylation by different kinases and by recruiting diverse E3 ubiquitin ligase to the receptor complex. Different proteolytic systems (ubiquitin-proteasome, calpains), chaperone activities and signaling pathways influence the stability of GRKs in different ways, thus endowing specificity to GPCR regulation as protein turnover of GRKs can be differentially affected. Therefore, modulation of protein stability of GRKs emerges as a versatile mechanism for feedback regulation of GPCR signaling and basic cellular processes. PMID:27378756

  12. 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...

  13. Down-regulation of protein kinase Ceta by antisense oligonucleotides sensitises A549 lung cancer cells to vincristine and paclitaxel.

    Science.gov (United States)

    Sonnemann, Jürgen; Gekeler, Volker; Ahlbrecht, Katrin; Brischwein, Klaus; Liu, Chao; Bader, Peter; Müller, Cornelia; Niethammer, Dietrich; Beck, James F

    2004-06-25

    Previous studies point to protein kinase C (PKC) isozyme eta as a resistance factor in cancer cells. Therefore, we investigated whether down-regulation of PKCeta with second generation antisense oligonucleotides (ODNs) would sensitise A549 human lung carcinoma cells to cytostatics. The effects were compared to the outcome of Bcl-xL down-regulation. Upon treatment with antisense ODNs, PKCeta and Bcl-xL were both significantly reduced on mRNA and protein level. Down-regulation of either PKCeta or Bcl-xL in combination with vincristine or paclitaxel resulted in a significant increase in caspase-3 activity compared to that in the control oligonucleotide treated cells. In addition, PKCeta down-regulation augmented vincristine-induced dissipation of mitochondrial transmembrane potential. In conclusion, these results confirm that PKCeta might represent a considerable resistance factor and an interesting target to improve anticancer chemotherapy. PMID:15159020

  14. Large-Scale Analysis of Kinase Signaling in Yeast Pseudohyphal Development Identifies Regulation of Ribonucleoprotein Granules.

    Directory of Open Access Journals (Sweden)

    Christian A Shively

    2015-10-01

    Full Text Available Yeast pseudohyphal filamentation is a stress-responsive growth transition relevant to processes required for virulence in pathogenic fungi. Pseudohyphal growth is controlled through a regulatory network encompassing conserved MAPK (Ste20p, Ste11p, Ste7p, Kss1p, and Fus3p, protein kinase A (Tpk2p, Elm1p, and Snf1p kinase pathways; however, the scope of these pathways is not fully understood. Here, we implemented quantitative phosphoproteomics to identify each of these signaling networks, generating a kinase-dead mutant in filamentous S. cerevisiae and surveying for differential phosphorylation. By this approach, we identified 439 phosphoproteins dependent upon pseudohyphal growth kinases. We report novel phosphorylation sites in 543 peptides, including phosphorylated residues in Ras2p and Flo8p required for wild-type filamentous growth. Phosphoproteins in these kinase signaling networks were enriched for ribonucleoprotein (RNP granule components, and we observe co-localization of Kss1p, Fus3p, Ste20p, and Tpk2p with the RNP component Igo1p. These kinases localize in puncta with GFP-visualized mRNA, and KSS1 is required for wild-type levels of mRNA localization in RNPs. Kss1p pathway activity is reduced in lsm1Δ/Δ and pat1Δ/Δ strains, and these genes encoding P-body proteins are epistatic to STE7. The P-body protein Dhh1p is also required for hyphal development in Candida albicans. Collectively, this study presents a wealth of data identifying the yeast phosphoproteome in pseudohyphal growth and regulatory interrelationships between pseudohyphal growth kinases and RNPs.

  15. NPM-ALK oncogenic tyrosine kinase controls T-cell identity by transcriptional regulation and epigenetic silencing in lymphoma cells.

    Science.gov (United States)

    Ambrogio, Chiara; Martinengo, Cinzia; Voena, Claudia; Tondat, Fabrizio; Riera, Ludovica; di Celle, Paola Francia; Inghirami, Giorgio; Chiarle, Roberto

    2009-11-15

    Transformed cells in lymphomas usually maintain the phenotype of the postulated normal lymphocyte from which they arise. By contrast, anaplastic large cell lymphoma (ALCL) is a T-cell lymphoma with aberrant phenotype because of the defective expression of the T-cell receptor and other T-cell-specific molecules for still undetermined mechanisms. The majority of ALCL carries the translocation t(2;5) that encodes for the oncogenic tyrosine kinase NPM-ALK, fundamental for survival, proliferation, and migration of transformed T cells. Here, we show that loss of T-cell-specific molecules in ALCL cases is broader than reported previously and involves most T-cell receptor-related signaling molecules, including CD3epsilon, ZAP70, LAT, and SLP76. We further show that NPM-ALK, but not the kinase-dead NPM-ALK(K210R), downregulated the expression of these molecules by a STAT3-mediated gene transcription regulation and/or epigenetic silencing because this downregulation was reverted by treating ALCL cells with 5-aza-2-deoxycytidine or by knocking down STAT3 through short hairpin RNA. Finally, NPM-ALK increased the methylation of ZAP70 intron 1-exon 2 boundary region, and both NPM-ALK and STAT3 regulated the expression levels of DNA methyltransferase 1 in transformed T cells. Thus, our data reveal that oncogene-deregulated tyrosine kinase activity controls the expression of molecules that determine T-cell identity and signaling.

  16. Intracellular transactivation of epidermal growth factor receptor by α1A-adrenoceptor is mediated by phosphatidylinositol 3-kinase independently of activation of extracellular signal regulated kinases 1/2 and serine-threonine kinases in Chinese hamster ovary cells.

    Science.gov (United States)

    Ulu, Nadir; Henning, Robert H; Guner, Sahika; Zoto, Teuta; Duman-Dalkilic, Basak; Duin, Marry; Gurdal, Hakan

    2013-10-01

    Transactivation of epidermal growth factor receptor (EGFR) by α1-adrenoceptor (α1-AR) is implicated in contraction and hypertrophy of vascular smooth muscle (VSM). We examine whether all α1-AR subtypes transactivate EGFR and explore the mechanism of transactivation. Chinese hamster ovary (CHO) cells stably expressing one subtype of α1-AR were transiently transfected with EGFR. The transactivation mechanism was examined both by coexpression of a chimeric erythropoietin (EPO)-EGFR with an extracellular EPO and intracellular EGFR domain, and by pharmacologic inhibition of external and internal signaling routes. All three α1-AR subtypes transactivated EGFR, which was dependent on the increase in intracellular calcium. The EGFR kinase inhibitor AG1478 [4-(3'-chloroanilino)-6,7-dimethoxyquinazoline] abrogated α1A-AR and α1D-AR induced phosphorylation of EGFR, but both the inhibition of matrix metalloproteinases by GM6001 [(R)-N4-hydroxy-N(1)-[(S)-2-(1H-indol-3-yl)-1-methylcarbamoyl-ethyl]-2-isobutyl-succinamide] or blockade of EGFR by cetuximab did not. Stimulation of α1A-AR and α1D-AR also induced phosphorylation of EPO-EGFR chimeric receptors. Moreover, α1A-AR stimulation enhanced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 and serine-threonine kinases (Akt), which were both unaffected by AG1478, indicating that ERK1/2 and Akt phosphorylation is independent of EGFR transactivation. Accordingly, inhibitors of ERK1/2 or Akt did not influence the α1A-AR-mediated EGFR transactivation. Inhibition of calcium/calmodulin-dependent kinase II (CaMKII), phosphatidylinositol 3-kinase (PI3K), and Src, however, did block EGFR transactivation by α1A-AR and α1D-AR. These findings demonstrate that all α1-AR subtypes transactivate EGFR, which is dependent on an intracellular signaling route involving an increase in calcium and activation of CaMKII, PI3K, and Src, but not the of ERK1/2 and Akt pathways.

  17. Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase

    International Nuclear Information System (INIS)

    Hypoxia has been considered to affect the properties of tissue stem cells including mesenchymal stem cells (MSCs). Effects of long periods of exposure to hypoxia on human MSCs, however, have not been clearly demonstrated. MSCs cultured under normoxic conditions (20% pO2) ceased to proliferate after 15-25 population doublings, while MSCs cultured under hypoxic conditions (1% pO2) retained the ability to proliferate with an additional 8-20 population doublings. Most of the MSCs cultured under normoxic conditions were in a senescent state after 100 days, while few senescent cells were found in the hypoxic culture, which was associated with a down-regulation of p16 gene expression. MSCs cultured for 100 days under hypoxic conditions were superior to those cultured under normoxic conditions in the ability to differentiate into the chondro- and adipogenic, but not osteogenic, lineage. Among the molecules related to mitogen-activated protein kinase (MAPK) signaling pathways, extracellular signal regulated kinase (ERK) was significantly down-regulated by hypoxia, which helped to inhibit the up-regulation of p16 gene expression. Therefore, the hypoxic culture retained MSCs in an undifferentiated and senescence-free state through the down-regulation of p16 and ERK.

  18. Protein kinase C is differentially regulated by thrombin, insulin, and epidermal growth factor in human mammary tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Gomez, M.L.; Tellez-Inon, M.T. (Instituto de Ingenieria Genetica y Biologia Molecular, Buenos Aires (Argentina)); Medrano, E.E.; Cafferatta, E.G.A. (Instituto de Investigaciones Bioquimicas Fundacion Campomar, Buenos Aires (Argentina))

    1988-03-01

    The exposure of serum-deprived mammary tumor cells MCF-7 and T-47D to insulin, thrombin, and epidermal growth factor (EGF) resulted in dramatic modifications in the activity and in the translocation capacity of protein kinase C from cytosol to membrane fractions. Insulin induces a 600% activation of the enzyme after 5 h of exposure to the hormone in MCF-7 cells; thrombin either activates (200% in MCF-7) or down-regulates (in T-47D), and EGF exerts only a moderate effect. Thus, the growth factors studied modulate differentially the protein kinase C activity in human mammary tumor cells. The physiological significance of the results obtained are discussed in terms of the growth response elicited by insulin, thrombin, and EGF.

  19. Regulation of the EphA2 kinase by the low molecular weight tyrosine phosphatase induces transformation.

    Science.gov (United States)

    Kikawa, Keith D; Vidale, Derika R; Van Etten, Robert L; Kinch, Michael S

    2002-10-18

    Intracellular signaling by protein tyrosine phosphorylation is generally understood to govern many aspects of cellular behavior. The biological consequences of this signaling pathway are important because the levels of protein tyrosine phosphorylation are frequently elevated in cancer cells. In the classic paradigm, tyrosine kinases promote tumor cell growth, survival, and invasiveness, whereas tyrosine phosphatases negatively regulate these same behaviors. Here, we identify one particular tyrosine phosphatase, low molecular weight tyrosine phosphatase (LMW-PTP), which is frequently overexpressed in transformed cells. We also show that overexpression of LMW-PTP is sufficient to confer transformation upon non-transformed epithelial cells. Notably, we show that the EphA2 receptor tyrosine kinase is a prominent substrate for LMW-PTP and that the oncogenic activities of LMW-PTP result from altered EphA2 expression and function. These results suggest a role for LMW-PTP in transformation progression and link its oncogenic potential to EphA2.

  20. A core of kinase-regulated interactomes defines the neoplastic MDSC lineage.

    Science.gov (United States)

    Gato-Cañas, Maria; Martinez de Morentin, Xabier; Blanco-Luquin, Idoia; Fernandez-Irigoyen, Joaquin; Zudaire, Isabel; Liechtenstein, Therese; Arasanz, Hugo; Lozano, Teresa; Casares, Noelia; Chaikuad, Apirat; Knapp, Stefan; Guerrero-Setas, David; Escors, David; Kochan, Grazyna; Santamaría, Enrique

    2015-09-29

    Myeloid-derived suppressor cells (MDSCs) differentiate from bone marrow precursors, expand in cancer-bearing hosts and accelerate tumor progression. MDSCs have become attractive therapeutic targets, as their elimination strongly enhances anti-neoplastic treatments. Here, immature myeloid dendritic cells (DCs), MDSCs modeling tumor-infiltrating subsets or modeling non-cancerous (NC)-MDSCs were compared by in-depth quantitative proteomics. We found that neoplastic MDSCs differentially expressed a core of kinases which controlled lineage-specific (PI3K-AKT and SRC kinases) and cancer-induced (ERK and PKC kinases) protein interaction networks (interactomes). These kinases contributed to some extent to myeloid differentiation. However, only AKT and ERK specifically drove MDSC differentiation from myeloid precursors. Interfering with AKT and ERK with selective small molecule inhibitors or shRNAs selectively hampered MDSC differentiation and viability. Thus, we provide compelling evidence that MDSCs constitute a distinct myeloid lineage distinguished by a "kinase signature" and well-defined interactomes. Our results define new opportunities for the development of anti-cancer treatments targeting these tumor-promoting immune cells. PMID:26320174

  1. Rewiring MAP kinases in Saccharomyces cerevisiae to regulate novel targets through ubiquitination.

    Science.gov (United States)

    Groves, Benjamin; Khakhar, Arjun; Nadel, Cory M; Gardner, Richard G; Seelig, Georg

    2016-01-01

    Evolution has often copied and repurposed the mitogen-activated protein kinase (MAPK) signaling module. Understanding how connections form during evolution, in disease and across individuals requires knowledge of the basic tenets that govern kinase-substrate interactions. We identify criteria sufficient for establishing regulatory links between a MAPK and a non-native substrate. The yeast MAPK Fus3 and human MAPK ERK2 can be functionally redirected if only two conditions are met: the kinase and substrate contain matching interaction domains and the substrate includes a phospho-motif that can be phosphorylated by the kinase and recruit a downstream effector. We used a panel of interaction domains and phosphorylation-activated degradation motifs to demonstrate modular and scalable retargeting. We applied our approach to reshape the signaling behavior of an existing kinase pathway. Together, our results demonstrate that a MAPK can be largely defined by its interaction domains and compatible phospho-motifs and provide insight into how MAPK-substrate connections form.

  2. The MAPKKK Ste11 regulates vegetative growth through a kinase cascade of shared signaling components.

    Science.gov (United States)

    Lee, B N; Elion, E A

    1999-10-26

    In haploid Saccharomyces cerevisiae, the mating and invasive growth (IG) pathways use the same mitogen-activated protein kinase kinase kinase kinase (MAPKKKK, Ste20), MAPKKK (Ste11), MAPKK (Ste7), and transcription factor (Ste12) to promote either G(1) arrest and fusion or foraging in response to distinct stimuli. This exquisite specificity is the result of pathway-specific receptors, G proteins, scaffold protein, and MAPKs. It is currently not thought that the shared signaling components function under the basal conditions of vegetative growth. We tested this hypothesis by searching for mutations that cause lethality when the STE11 gene is deleted. Strikingly, we found that Ste11, together with Ste20, Ste7, Ste12, and the IG MAPK Kss1, functions in a third pathway that promotes vegetative growth and is essential in an och1 mutant that does not synthesize mannoproteins. We term this pathway the STE vegetative growth (SVG) pathway. The SVG pathway functions, in part, to promote cell wall integrity in parallel with the protein kinase C pathway. During vegetative growth, the SVG pathway is inhibited by the mating MAPK Fus3. By contrast, the SVG pathway is constitutively activated in an och1 mutant, suggesting that it senses intracellular changes arising from the loss of mannoproteins. We predict that general proliferative functions may also exist for other MAPK cascades thought only to perform specialized functions. PMID:10535982

  3. Comparative proteomics of a tor inducible Aspergillus fumigatus mutant reveals involvement of the Tor kinase in iron regulation.

    Science.gov (United States)

    Baldin, Clara; Valiante, Vito; Krüger, Thomas; Schafferer, Lukas; Haas, Hubertus; Kniemeyer, Olaf; Brakhage, Axel A

    2015-07-01

    The Tor (target of rapamycin) kinase is one of the major regulatory nodes in eukaryotes. Here, we analyzed the Tor kinase in Aspergillus fumigatus, which is the most important airborne fungal pathogen of humans. Because deletion of the single tor gene was apparently lethal, we generated a conditional lethal tor mutant by replacing the endogenous tor gene by the inducible xylp-tor gene cassette. By both 2DE and gel-free LC-MS/MS, we found that Tor controls a variety of proteins involved in nutrient sensing, stress response, cell cycle progression, protein biosynthesis and degradation, but also processes in mitochondria, such as respiration and ornithine metabolism, which is required for siderophore formation. qRT-PCR analyses indicated that mRNA levels of ornithine biosynthesis genes were increased under iron limitation. When tor was repressed, iron regulation was lost. In a deletion mutant of the iron regulator HapX also carrying the xylp-tor cassette, the regulation upon iron deprivation was similar to that of the single tor inducible mutant strain. In line, hapX expression was significantly reduced when tor was repressed. Thus, Tor acts either upstream of HapX or independently of HapX as a repressor of the ornithine biosynthesis genes and thereby regulates the production of siderophores.

  4. Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

    Energy Technology Data Exchange (ETDEWEB)

    Severson, Eric A.; Kwon, Mike; Hilgarth, Roland S.; Parkos, Charles A. [Epithelial Pathobiology Research Unit, Dept. of Pathology, Emory University, Atlanta, GA 30322 (United States); Nusrat, Asma, E-mail: anusrat@emory.edu [Epithelial Pathobiology Research Unit, Dept. of Pathology, Emory University, Atlanta, GA 30322 (United States)

    2010-07-02

    The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors or siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.

  5. Rho A and the Rho kinase pathway regulate fibroblast contraction: Enhanced contraction in constitutively active Rho A fibroblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nobe, Koji, E-mail: kojinobe@pharm.showa-u.ac.jp [Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo (Japan); Nobe, Hiromi [Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo (Japan); Department of Physical Therapy, Bunkyo-Gakuin University (Japan); Yoshida, Hiroko [Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo (Japan); Kolodney, Michael S. [Dermatology Division, Department of Medicine, UCLA, Los Angeles, CA (United States); Paul, Richard J. [Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, OH (United States); Honda, Kazuo [Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo (Japan)

    2010-08-20

    Research highlights: {yields} Mechanisms of fibroblast cell contraction in collagen matrix. {yields} Assessed an isometric force development using 3D-reconstituted-fibroblast fiber. {yields} Constitutively active Rho A induced the over-contraction of fibroblast cells. {yields} Rho A and Rho kinase pathway has a central role in fibroblast cell contraction. -- Abstract: Fibroblast cells play a central role in the proliferation phase of wound healing processes, contributing to force development. The intracellular signaling pathways regulating this non-muscle contraction are only partially understood. To study the relations between Rho A and contractile responses, constitutively active Rho A (CA-Rho A) fibroblast cells were reconstituted into fibers and the effects of calf serum (CS) on isometric force were studied. CS-induced force in CA-Rho A fibroblast fibers was twice as large as that in wild type (NIH 3T3) fibroblast fibers. During this response, the translocation of Rho A from the cytosol to the membrane was detected by Rho A activity assays and Western blot analysis. Pre-treatment with a Rho specific inhibitor (C3-exoenzyme) suppressed translocation as well as contraction. These results indicate that Rho A activation is essential for fibroblast contraction. The Rho kinase inhibitor ( (Y27632)) inhibited both NIH 3T3 and CA-Rho A fibroblast fiber contractions. Activation of Rho A is thus directly coupled with Rho kinase activity. We conclude that the translocation of Rho A from the cytosol to the membrane and the Rho kinase pathway can regulate wound healing processes mediated by fibroblast contraction.

  6. Role of a novel PH-kinase domain interface in PKB/Akt regulation: structural mechanism for allosteric inhibition.

    Directory of Open Access Journals (Sweden)

    Véronique Calleja

    2009-01-01

    Full Text Available Protein kinase B (PKB/Akt belongs to the AGC superfamily of related serine/threonine protein kinases. It is a key regulator downstream of various growth factors and hormones and is involved in malignant transformation and chemo-resistance. Full-length PKB protein has not been crystallised, thus studying the molecular mechanisms that are involved in its regulation in relation to its structure have not been simple. Recently, the dynamics between the inactive and active conformer at the molecular level have been described. The maintenance of PKB's inactive state via the interaction of the PH and kinase domains prevents its activation loop to be phosphorylated by its upstream activator, phosphoinositide-dependent protein kinase-1 (PDK1. By using a multidisciplinary approach including molecular modelling, classical biochemical assays, and Förster resonance energy transfer (FRET/two-photon fluorescence lifetime imaging microscopy (FLIM, a detailed model depicting the interaction between the different domains of PKB in its inactive conformation was demonstrated. These findings in turn clarified the molecular mechanism of PKB inhibition by AKT inhibitor VIII (a specific allosteric inhibitor and illustrated at the molecular level its selectivity towards different PKB isoforms. Furthermore, these findings allude to the possible function of the C-terminus in sustaining the inactive conformer of PKB. This study presents essential insights into the quaternary structure of PKB in its inactive conformation. An understanding of PKB structure in relation to its function is critical for elucidating its mode of activation and discovering how to modulate its activity. The molecular mechanism of inhibition of PKB activation by the specific drug AKT inhibitor VIII has critical implications for determining the mechanism of inhibition of other allosteric inhibitors and for opening up opportunities for the design of new generations of modulator drugs.

  7. Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3β.

    Science.gov (United States)

    Wang, Shuxin; Li, Zheyi; Shen, Hongyan; Zhang, Zhong; Yin, Yuxin; Wang, Qingsong; Zhao, Xuyang; Ji, Jianguo

    2016-08-01

    Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation. More than 65% of class I phosphosites were novel when compared with PhosphoSitePlus database. Quantification results showed that the early and late stage of NSC differentiation differ greatly. We mapped 69 changed phosphosites on 20 proteins involved in Wnt signaling pathway, including S552 on catenin beta-1 (Ctnnb1) and S9 on glycogen synthase kinase 3β (Gsk3β). Western blotting and real-time PCR results proved that Wnt signaling pathway plays critical roles in NSC fate determination. Furthermore, inhibition and activation of PKA dramatically affected the phosphorylation state of Ctnnb1 and Gsk3β, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self-renewal and differentiation of NSCs. Stem Cells 2016;34:2090-2101. PMID:27097102

  8. Role of a mitogen-activated protein kinase cascade in ion flux-mediated turgor regulation in fungi.

    Science.gov (United States)

    Lew, Roger R; Levina, Natalia N; Shabala, Lana; Anderca, Marinela I; Shabala, Sergey N

    2006-03-01

    Fungi normally maintain a high internal hydrostatic pressure (turgor) of about 500 kPa. In response to hyperosmotic shock, there are immediate electrical changes: a transient depolarization (1 to 2 min) followed by a sustained hyperpolarization (5 to 10 min) prior to turgor recovery (10 to 60 min). Using ion-selective vibrating probes, we established that the transient depolarization is due to Ca(2+) influx and the sustained hyperpolarization is due to H(+) efflux by activation of the plasma membrane H(+)-ATPase. Protein synthesis is not required for H(+)-ATPase activation. Net K(+) and Cl(-) uptake occurs at the same time as turgor recovery. The magnitude of the ion uptake is more than sufficient to account for the osmotic gradients required for turgor to return to its original level. Two osmotic mutants, os-1 and os-2, homologs of a two-component histidine kinase sensor and the yeast high osmotic glycerol mitogen-activated protein (MAP) kinase, respectively, have lower turgor than the wild type and do not exhibit the sustained hyperpolarization after hyperosmotic treatment. The os-1 mutant does not exhibit all of the wild-type turgor-adaptive ion fluxes (Cl(-) uptake increases, but net K(+) flux barely changes and net H(+) efflux declines) (os-2 was not examined). Both os mutants are able to regulate turgor but at a lower level than the wild type. Our results demonstrate that a MAP kinase cascade regulates ion transport, activation of the H(+)-ATPase, and net K(+) and Cl(-) uptake during turgor regulation. Other pathways regulating turgor must also exist. PMID:16524903

  9. Role of a Mitogen-Activated Protein Kinase Cascade in Ion Flux-Mediated Turgor Regulation in Fungi

    Science.gov (United States)

    Lew, Roger R.; Levina, Natalia N.; Shabala, Lana; Anderca, Marinela I.; Shabala, Sergey N.

    2006-01-01

    Fungi normally maintain a high internal hydrostatic pressure (turgor) of about 500 kPa. In response to hyperosmotic shock, there are immediate electrical changes: a transient depolarization (1 to 2 min) followed by a sustained hyperpolarization (5 to 10 min) prior to turgor recovery (10 to 60 min). Using ion-selective vibrating probes, we established that the transient depolarization is due to Ca2+ influx and the sustained hyperpolarization is due to H+ efflux by activation of the plasma membrane H+-ATPase. Protein synthesis is not required for H+-ATPase activation. Net K+ and Cl− uptake occurs at the same time as turgor recovery. The magnitude of the ion uptake is more than sufficient to account for the osmotic gradients required for turgor to return to its original level. Two osmotic mutants, os-1 and os-2, homologs of a two-component histidine kinase sensor and the yeast high osmotic glycerol mitogen-activated protein (MAP) kinase, respectively, have lower turgor than the wild type and do not exhibit the sustained hyperpolarization after hyperosmotic treatment. The os-1 mutant does not exhibit all of the wild-type turgor-adaptive ion fluxes (Cl− uptake increases, but net K+ flux barely changes and net H+ efflux declines) (os-2 was not examined). Both os mutants are able to regulate turgor but at a lower level than the wild type. Our results demonstrate that a MAP kinase cascade regulates ion transport, activation of the H+-ATPase, and net K+ and Cl− uptake during turgor regulation. Other pathways regulating turgor must also exist. PMID:16524903

  10. Atorvastatin induces autophagy of mesenchymal stem cells under hypoxia and serum deprivation conditions by activating the mitogenactivated protein kinase/extracellular signal-regulated kinase pathway

    Institute of Scientific and Technical Information of China (English)

    Li Na; Zhang Qian; Qian Haiyan; Jin Chen; Yang Yuejin; Gao Runlin

    2014-01-01

    Background The survival ratio of implanted mesenchymal stem cells (MSCs) in the infarcted myocardium is low.Autophagy is a complex "self-eating" process and can be utilized for cell survival.We have found that atorvastatin (ATV) can effectively activate autophagy to enhance MSCs survival during hypoxia and serum deprivation (H/SD).The mitogenactivated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway is a non-canonical autophagy pathway.We hypothesized that the MEK/ERK pathway mediated ATV-induced autophagy of MSCs under H/SD.Methods MSCs were pretreated with ATV (0.01-10 μmol/L) under H/SD for three hours.For inhibitor studies,the cells were pre-incubated with the MEK1/2 inhibitor U0126.Cell autophagy was assessed by acidic vesicular organelles (AVO)-positive cells using flow cytometry,autophagy related protein using Western blotting and autophagosome using transmission electron microscopy.Results Autophagy was elevated in the H/SD group compared with the normal group.ATV further enhanced the autophagic activity as well as the phosphorylation of ERK1/2 evidenced by more AVO-positive cells ((8.63±0.63)% vs.(5.77±0.44)%,P <0.05),higher LC3-Ⅱ/LC3-Ⅰ ratio (4.36±0.31 vs.2.52±0.18,P <0.05) and more autophagosomes.And treatment with U0126 downregulated the phosphorylation of ERK1/2 and attenuated ATV-induced autophagy.Conclusion The MEK/ERK pathway participates in ATV-induced autophagy in MSCs under H/SD,and modulation of the pathway could be a novel strategy to improve MSCs survival.

  11. Imbalanced expression of mitogen-activated protein kinase phosphatase-1 and phosphorylated extracellular signal-regulated kinases in lung squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Kai WANG; Min ZHANG; Ying-ying QIAN; Zhe-yuan DING; Jun-huiLV; Hua-hao SHEN

    2011-01-01

    Objective:Mitogen-activated protein kinases (MAPKs) are correlated with a more malignant phenotype in many cancers.This study was designed to evaluate the predictive value of the expression of MAPK phosphatase-1 (MKP-1) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2),as the key regulatory mechanism of the MAPKs,in lung squamous cell carcinoma (SCC).Methods:We assessed the expressions of MKP-1 and p-ERK1/2in twenty subjects at different differentiation degree of SCC and five normal lungs by immunohistochemistry and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis.Results:Immunohistochemistry and real-time RT-PCR assay showed that the expression of MKP-1 was gradually decreased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was negatively correlated with tumor differentiation (P<0.01).However,the expression of p-ERK1/2 or ERK1/2 was gradually increased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was positively correlated with tumor differentiation (P<0.01).Conclusions:Our data indicates the relevance of MKP-1 and p-ERK1/2 in SCC as a potential positive and negative prognostic factor.The imbalanced expression of MKP-1 and p-ERK1/2 may play a role in the development of SCC and these two molecules may be new targets for the therapy and prognosis of SCC.

  12. Structural basis for substrate specificities of cellular deoxyribonucleoside kinases

    DEFF Research Database (Denmark)

    Johansson, K.; Ramaswamy, S.; Ljungcrantz, C.;

    2001-01-01

    kinase with ATP at the nucleoside substrate binding site. Compared to the human kinase, the Drosophila kinase has a wider substrate cleft, which may be responsible for the broad substrate specificity of this enzyme. The human deoxyguanosine kinase is highly specific for purine substrates......; this is apparently due to the presence of Arg 118, which provides favorable hydrogen bonding interactions with the substrate. The two new structures provide an explanation for the substrate specificity of cellular deoxyribonucleoside kinases....

  13. GPCR kinase 2 interacting protein 1 (GIT1) regulates osteoclast function and bone mass

    OpenAIRE

    Menon, Prashanthi; Yin, Guoyong; Smolock, Elaine M.; Zuscik, Michael J.; Yan, Chen; Berk, Bradford C.

    2010-01-01

    G-protein coupled receptor (GPCR) kinase 2 interacting protein-1 (GIT1) is a scaffold protein expressed in various cell types including neurons, endothelial and vascular smooth muscle cells. The GIT1 knockout (KO) mouse has a pulmonary phenotype due to impaired endothelial function. Because GIT1 is tyrosine phosphorylated by Src kinase, we anticipated that GIT1 KO should have a bone phenotype similar to Src KO. Microcomputed tomography of the long bones revealed that GIT1 KO mice have a 2.3-f...

  14. Ability of CK2beta to selectively regulate cellular protein kinases

    DEFF Research Database (Denmark)

    Olsen, Birgitte; Guerra, Barbara

    2008-01-01

    The Wee1 protein kinase plays a prominent role in keeping cyclin dependent kinase 1 (CDK1) inactive during the G2 phase of the cell cycle. At the onset of mitosis, Wee1 is ubiquitinated by the E3 ubiquitin ligase SCF(beta-TrCP) and subsequently degraded by the proteasome machinery. Previously...... additional phosphodegrons recognised by beta-TrCP. These events contribute to destabilise Wee1 at the onset of mitosis (Watanabe et al. Proc Natl Acad Sci USA 101:4419-4424, 2004). We show here that in addition to the ability of CK2 to phosphorylate Wee1 as reported earlier, the regulatory beta...

  15. Janus kinase 2 regulates Bcr–Abl signaling in chronic myeloid leukemia

    OpenAIRE

    Samanta, A.; Perazzona, B; Chakraborty, S.; Sun, X.; Modi, H; Bhatia, R.; Priebe, W.; Arlinghaus, R.

    2010-01-01

    Despite the success of imatinib mesylate (IM) in the early chronic phase of chronic myeloid leukemia (CML), patients are resistant to IM and other kinase inhibitors in the later stages of CML. Our findings indicate that inhibition of Janus kinase 2 (Jak2) in Bcr–Abl+ cells overcomes IM resistance although the precise mechanism of Jak2 action is unknown. Knocking down Jak2 in Bcr–Abl+ cells reduced levels of the Bcr–Abl protein and also the phosphorylation of Tyr177 of Bcr–Abl, and Jak2 overex...

  16. Budding yeast PAK kinases regulate mitotic exit by two different mechanisms

    OpenAIRE

    Chiroli, Elena; Fraschini, Roberta; Beretta, Alessia; Tonelli, Mariagrazia; Lucchini, Giovanna; Piatti, Simonetta

    2003-01-01

    We report the characterization of the dominant-negative CLA4t allele of the budding yeast CLA4 gene, encoding a member of the p21-activated kinase (PAK) family of protein kinases, which, together with its homologue STE20, plays an essential role in promoting budding and cytokinesis. Overproduction of the Cla4t protein likely inhibits both endogenous Cla4 and Ste20 and causes a delay in the onset of anaphase that correlates with inactivation of Cdc20/anaphase-promoting complex (APC)–dependent ...

  17. Protein kinase C (PKC) alpha and PKC theta are the major PKC isotypes involved in TCR down-regulation

    DEFF Research Database (Denmark)

    von Essen, Marina; Nielsen, Martin W; Bonefeld, Charlotte M;

    2006-01-01

    It is well known that protein kinase C (PKC) plays an important role in regulation of TCR cell surface expression levels. However, eight different PKC isotypes are present in T cells, and to date the particular isotype(s) involved in TCR down-regulation remains to be identified. The aim...... of this study was to identify the PKC isotype(s) involved in TCR down-regulation and to elucidate the mechanism by which they induce TCR down-regulation. To accomplish this, we studied TCR down-regulation in the human T cell line Jurkat, in primary human T cells, or in the mouse T cell line DO11.10 in which we...... to induce significant TCR down-regulation. Both isotypes mediated TCR down-regulation via the TCR recycling pathway that strictly depends on Ser(126) and the di-leucine-based receptor-sorting motif of the CD3gamma chain. Finally, we found that PKCtheta was mainly implicated in down-regulation of directly...

  18. Protein kinase Cθ gene expression is oppositely regulated by GCN5 and EBF1 in immature B cells.

    Science.gov (United States)

    Kikuchi, Hidehiko; Nakayama, Masami; Kuribayashi, Futoshi; Imajoh-Ohmi, Shinobu; Nishitoh, Hideki; Takami, Yasunari; Nakayama, Tatsuo

    2014-05-01

    In this study, we revealed that GCN5 and early B cell factor 1 (EBF1) participate in regulation of protein kinase Cθ (PKCθ) gene expression in an opposite manner in immature B cells. GCN5-deficiency in DT40 caused drastic down-regulation of transcription of PKCθ. In contrast, EBF1-deficiency brought about remarkable up-regulation of that of PKCθ, and re-expression of EBF1 dramatically suppressed transcription of PKCθ. Chromatin immunoprecipitation assay revealed that GCN5 binds to the 5'-flanking region of the chicken PKCθ gene and acetylates histone H3, and EBF1 binds to the 5'-flanking region of the gene surrounding putative EBF1 binding motifs.

  19. Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2 alpha (eIF-2 alpha) kinase of rabbit reticulocytes: homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2 alpha kinase.

    Science.gov (United States)

    Chen, J J; Throop, M S; Gehrke, L; Kuo, I; Pal, J K; Brodsky, M; London, I M

    1991-01-01

    We have cloned the cDNA of the heme-regulated eIF-2 alpha kinase (HRI) of rabbit reticulocytes. In vitro translation of mRNA transcribed from the HRI cDNA yields a 90-kDa polypeptide that exhibits eIF-2 alpha kinase activity and is recognized by a monoclonal antibody directed against authentic HRI. The open reading frame sequence of the HRI cDNA contains all 11 catalytic domains of protein kinases with consensus sequences of protein-serine/threonine kinases in conserved catalytic domains VI and VIII. The HRI cDNA also contains an insert of approximately 140 amino acids between catalytic domains V and VI. The HRI cDNA coding sequence has extensive homology to GCN2 protein kinase of Saccharomyces cerevisiae and to human double-stranded-RNA-dependent eIF-2 alpha kinase. This observation suggests that GCN2 protein kinase may be an eIF-2 alpha kinase in yeast. In addition, HRI has an unusually high degree of homology to three protein kinases (NimA, Wee1, and CDC2) that are involved in the regulation of the cell cycle. Images PMID:1679235

  20. Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2. alpha. (eIF-2. alpha. ) kinase of rabbit reticulocytes: Homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2. alpha. kinase

    Energy Technology Data Exchange (ETDEWEB)

    Chen, J.J.; Throop, M.S.; Kuo, I.; Pal, J.K.; Brodsky, M.; London, I.M. (Massachusetts Inst. of Tech., Cambridge (United States)); Gehrke, L. (Massachusetts Inst. of Tech., Cambridge (United States) Harvard Medical School, Boston, MA (United States))

    1991-09-01

    The authors have cloned the cDNA of the heme-regulated eIF-2{alpha} kinase (HRI) of rabbit reticulocytes. In vitro translation of mRNA transcribed from the HRI cDNA yields a 90-kDa polypeptide that exhibits eIF-2{alpha} kinase activity and is recognized by a monoclonal antibody directed against authentic HRI. The open reading frame sequence of the HRI cDNA contains all 11 catalytic domains of protein kinases with consensus sequences of protein-serine/threonine kinases in conserved catalytic domains VI and VIII. The HRI cDNA also contains an insert of {approx} 140 amino acids between catalytic domains V and VI. The HRI cDNA coding sequence has extensive homology to GCN2 protein kinase of Saccharomyces cerevisiae and to human double-stranded-RNA-dependent eIF-2{alpha} kinase. This observation suggests that GCN2 protein kinase may be an eIF-2{alpha} kinase in yeast. In addition, HRI has an unusually high degree of homology to three protein kinases (NimA, Wee1, and CDC2) that are involved in the regulation of the cell cycle.

  1. 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

  2. The AMPK-related kinase SNARK regulates muscle mass and myocyte survival

    Science.gov (United States)

    The maintenance of skeletal muscle mass is critical for sustaining health; however, the mechanisms responsible for muscle loss with aging and chronic diseases, such as diabetes and obesity, are poorly understood. We found that expression of a member of the AMPK-related kinase family, the SNF1-AMPK-r...

  3. Regulation of taurine transport systems by protein kinase CK2 in mammalian cells

    DEFF Research Database (Denmark)

    Lambert, Ian Henry; Hansen, Daniel Bloch

    2011-01-01

    Maintaining cell volume is critical for cellular function yet shift in cell volume is a prerequisite for mitosis and apoptosis. The ubiquitously and evolutionary conserved serine/threonine kinase CK2 promotes cell survival and suppresses apoptosis. The present review describes how mammalian cells...

  4. Regulation of taurine homeostasis by protein kinase CK2 in mouse fibroblasts

    DEFF Research Database (Denmark)

    Hansen, Daniel Bloch; Guerra, Barbara; Jacobsen, Jack Hummeland;

    2011-01-01

    Increased expression of the ubiquitous serine/threonine protein kinase CK2 has been associated with increased proliferative capacity and increased resistance towards apoptosis. Taurine is the primary organic osmolyte involved in cell volume control in mammalian cells, and shift in cell volume is ...

  5. Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus

    DEFF Research Database (Denmark)

    Olivares-Illana, Vanesa; Meyer, Philippe; Bechet, Emmanuelle;

    2008-01-01

    understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus...

  6. Syndecan-4 proteoglycan regulates the distribution and activity of protein kinase C

    DEFF Research Database (Denmark)

    Oh, E S; Woods, A; Couchman, J R

    1997-01-01

    During cell-matrix adhesion, both tyrosine and serine/threonine kinases are activated. Integrin ligation correlates with tyrosine phosphorylation, whereas the later stages of spreading and focal adhesion and stress fiber formation in primary fibroblasts requires interactions of cell surface...... adhesions. This represents the first report of direct transmembrane signaling through cell surface proteoglycans....

  7. Regulation of salt and ABA responses by CIPK14, a calcium sensor interacting protein kinase in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Calcium and protein kinase serve as the common mediators to regulate plant responses to multiple stresses including salt and ABA stimulus. Here we reported a novel protein kinase (CIPK14) that regulated the responses to ABA treatment and salt stress in Arabidopsis. CIPK14 transcripts, capable been checked in roots, stems, leaves and flowers, were highly expressed in flowers and roots. CIPK14 was induced by ABA and salt treatments. The disruption of CIPK14 altered the transcriptional pattern of a gene marker line related to ABA and salt responses, and the results suggested that CIPK14 probably was responsible to the control of the salt and ABA responses. Comparing with wild types, the lines inserted with the T-DNA in which CIPK14 gene expression was knocked out were also more sensitive to ABA and salt stimulus, showing low germination rate and the less root elongation. While, when these conditioned seeds were treated with norflurazon, their germination percentages could recover to a certain extent. We also found that exogenous calcium could have an effect on the transcription of CIPK14 under ABA and salt treatments, and it seemed that calcium ion might work upstream CIPK14 to regulate the plant response to ABA and salt response.

  8. Dual Regulation of a Chimeric Plant Serine/Threonine Kinase by Calcium and Calcium/Calmodulin

    Science.gov (United States)

    Takezawa, D.; Ramachandiran, S.; Paranjape, V.; Poovaiah, B. W.

    1996-01-01

    A chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) gene characterized by a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain was recently cloned from plants. The Escherichia coli-expressed CCaMK phosphorylates various protein and peptide substrates in a Ca(2+)/calmodulin-dependent manner. The calmodulin-binding region of CCAMK has similarity to the calmodulin-binding region of the alpha-subunit of multifunctional Ca(2+)/calmodulin-dependent protein kinase (CaMKII). CCaMK exhibits basal autophosphorylation at the threonine residue(s) (0.098 mol of P-32/mol) that is stimulated 3.4-fold by Ca(2+) (0.339 mol of P-32/mol), while calmodulin inhibits Ca(2+)-stimulated autophosphorylation to the basal level. A deletion mutant lacking the visinin-like domain did not show Ca(2+)-simulated autophosphorylation activity but retained Ca(2+)/calmodulin-dependent protein kinase activity at a reduced level. Ca(2+)-dependent mobility shift assays using E.coli-expressed protein from residues 358-520 revealed that Ca(2+) binds to the visinin-like domain. Studies with site-directed mutants of the visinin-like domain indicated that EF-hands II and III are crucial for Ca(2+)-induced conformational changes in the visinin-like domain. Autophosphorylation of CCaMK increases Ca(2+)/calmodulin-dependent protein kinase activity by about 5-fold, whereas it did not affect its C(2+)-independent activity. This report provides evidence for the existence of a protein kinase in plants that is modulated by Ca(2+) and Ca(2+)/calmodulin. The presence of a visinin-like Ca(2+)-binding domain in CCaMK adds an additional Ca(2+)-sensing mechanism not previously known to exist in the Ca(2+)/calmodulin-mediated signaling cascade in plants.

  9. Receptor-Like Kinase RUPO Interacts with Potassium Transporters to Regulate Pollen Tube Growth and Integrity in Rice

    Science.gov (United States)

    Liu, Lingtong; Zheng, Canhui; Kuang, Baijan; Wei, Liqin; Yan, Longfeng; Wang, Tai

    2016-01-01

    During sexual reproduction of flowering plants, the pollen tube grows fast and over a long distance within the pistil to deliver two sperms for double fertilization. Growing plant cells need to communicate constantly with external stimuli as well as monitor changes in surface tension of the cell wall and plasma membrane to coordinate these signals and internal growth machinery; however, the underlying mechanisms remain largely unknown. Here we show that the rice member of plant-specific receptor-like kinase CrRLK1Ls subfamily, Ruptured Pollen tube (RUPO), is specifically expressed in rice pollen. RUPO localizes to the apical plasma membrane and vesicle of pollen tubes and is required for male gamete transmission. K+ levels were greater in pollen of homozygous CRISPR-knockout lines than wild-type plants, and pollen tubes burst shortly after germination. We reveal the interaction of RUPO with high-affinity potassium transporters. Phosphorylation of RUPO established and dephosphorylation abolished the interaction. These results have revealed the receptor-like kinase as a regulator of high-affinity potassium transporters via phosphorylation-dependent interaction, and demonstrated a novel receptor-like kinase signaling pathway that mediates K+ homeostasis required for pollen tube growth and integrity. PMID:27447945

  10. Induction of interleukin-8 by Naegleria fowleri lysates requires activation of extracellular signal-regulated kinase in human astroglial cells.

    Science.gov (United States)

    Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Sang-Hee; Kwon, Daeho; Shin, Ho-Joon

    2012-08-01

    Naegleria fowleri is a pathogenic free-living amoeba which causes primary amoebic meningoencephalitis in humans and experimental animals. To investigate the mechanisms of such inflammatory diseases, potential chemokine gene activation in human astroglial cells was investigated following treatment with N. fowleri lysates. We demonstrated that N. fowleri are potent inducers for the expression of interleukin-8 (IL-8) genes in human astroglial cells which was preceded by activation of extracellular signal-regulated kinase (ERK). In addition, N. fowleri lysates induces the DNA binding activity of activator protein-1 (AP-1), an important transcription factor for IL-8 induction. The specific mitogen-activated protein kinase kinase/ERK inhibitor, U0126, blocks N. fowleri-mediated AP-1 activation and subsequent IL-8 induction. N. fowleri-induced IL-8 expression requires activation of ERK in human astroglial cells. These findings indicate that treatment of N. fowleri on human astroglial cells leads to the activation of AP-1 and subsequent expression of IL-8 which are dependent on ERK activation. These results may help understand the N. fowleri-mediated upregulation of chemokine and cytokine expression in the astroglial cells.

  11. Regulation of EGF-induced ERK/MAPK Activation and EGFR Internalization by G Protein-coupled Receptor Kinase 2

    Institute of Scientific and Technical Information of China (English)

    Jingxia GAO; Jiali LI; Lan MA

    2005-01-01

    G protein-coupled receptor kinases (GRKs) mediate agonist-induced phosphorylation and desensitization of various G protein-coupled receptors (GPCRs). We investigate the role of GRK2 on epidermal growth factor (EGF) receptor signaling, including EGF-induced extracellular signal-regulated kinase and mitogen-activated protein kinase (ERK/MAPK) activation and EGFR internalization. Immunoprecipitation and immunofluorescence experiments show that EGF stimulates GRK2 binding to EGFR complex and GRK2 translocating from cytoplasm to the plasma membrane in human embryonic kidney 293 cells. Western blotting assay shows that EGF-induced ERK/MAPK phosphorylation increases 1.9-fold, 1.1-fold and 1.5-fold (P<0.05) at time point 30, 60 and 120 min, respectively when the cells were transfected with GRK2,suggesting the regulatory role of GRK2 on EGF-induced ERK/MAPK activation. Flow cytometry experiments show that GRK2 overexpression has no effect on EGF-induced EGFR internalization, however, it increases agonist-induced G protein-coupled δ opioid receptor internalization by approximately 40% (P<0.01). Overall,these data suggest that GRK2 has a regulatory role in EGF-induced ERK/MAPK activation, and that the mechanisms underlying the modulatory role of GRK2 in EGFR and GPCR signaling pathways are somewhat different at least in receptor internalization.

  12. The role(s) of Src kinase and Cbl proteins in the regulation of osteoclast differentiation and function.

    Science.gov (United States)

    Horne, William C; Sanjay, Archana; Bruzzaniti, Angela; Baron, Roland

    2005-12-01

    The osteoclast resorbs mineralized bone during bone development, homeostasis, and repair. The deletion of the gene encoding the nonreceptor tyrosine kinase c-Src produces an osteopetrotic skeletal phenotype that is the consequence of the inability of the mature osteoclast to efficiently resorb bone. Src-/- osteoclasts exhibit reduced motility and abnormal organization of the apical secretory domain (the ruffled border) and attachment-related cytoskeletal elements that are necessary for bone resorption. A key function of Src in osteoclasts is to promote the rapid assembly and disassembly of the podosomes, the specialized integrin-based attachment structures of osteoclasts and other highly motile cells. Once recruited to the activated integrins, especially alphavbeta3), by the adhesion tyrosine kinase Pyk2, Src binds and phosphorylates Cbl and Cbl-b, homologous multisite adapter proteins with ubiquitin ligase activity. The Cbl proteins in turn recruit and activate additional signaling effectors, including phosphatidylinositol 3-kinase and dynamin, which play key roles in the development of cell polarity and the regulation of cell attachment and motility. In addition, Src and the Cbl proteins contribute to signaling cascades that are activated by several important receptors, including receptor activator of nuclear factor kappaB and the macrophage colony-stimulating factor receptor, and also downregulate the signaling from many of these receptors. PMID:16313344

  13. Association between p21 Ser31Arg polymorphism and cancer risk: a meta-analysis

    Institute of Scientific and Technical Information of China (English)

    Hongxia Ma; Ziyuan Zhou; Sheng Wei; Qingyi Wei

    2011-01-01

    P21 (CDKN1A), a key cell cycle regulatory protein that governs cell cycle progression from G1 to S phase, can regulate cell proliferation, growth arrest, and apoptosis. The Ser31Arg polymorphism is located in the highly conserved region of p21 and may encode functionally distinct proteins. Although many epidemiological studies have been conducted to evaluate the association between the p21 Ser31Arg polymorphism and cancer risk, the findings remain conflicting. This meta-analysis with 33 077 cases and 45 013 controls from 44 published case-control studies showed that the variant homozygous 31Arg/Arg genotype was associated with an increased risk of numerous types of cancers in a random-effect model (homozygote comparison: OR = 1.17, 95% CI = 0.99 to 1.37, P = 0.0002 for the heterogeneity test; recessive model comparison: OR = 1.16, 95% CI = 1.01 to 1.33, P = 0.0001 for the heterogeneity test). Stratified analysis revealed that increased cancer risk associated with the 31Arg/Arg genotype remained significant in subgroups of colorectal cancer, estrogen-related cancer, Caucasians, population-based studies, studies with matching information or a larger sample size. Heterogeneity analysis showed that tumor type contributed to substantial between-study heterogeneity (recessive model comparison: x2 = 21.83, df = 7, P = 0.003). The results from this large-sample sized meta-analysis suggest that the p21 31Arg/Arg genotype may serve as a potential marker for increased cancer risk.

  14. Phosphatidylinositol 3-Kinase Plays a Vital Role in Regulation of Rice Seed Vigor via Altering NADPH Oxidase Activity

    OpenAIRE

    Jian Liu; Jun Zhou; Da Xing

    2012-01-01

    Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was al...

  15. Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Joanne I.; Kettering, Regina; Saxl, Ruth; Bourand, Alexa; Darbon, Emmanuelle; Joly, Nathalie; Briozzo, Pierre; Deutscher, Josef; (Pitt); (CNRS-CRMD)

    2009-09-11

    Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft are more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ('thermal factors') in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of

  16. AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human platelets.

    Science.gov (United States)

    Fleming, Ingrid; Schulz, Christian; Fichtlscherer, Birgit; Kemp, Bruce E; Fisslthaler, Beate; Busse, Rudi

    2003-11-01

    Little is known about the signaling cascades that eventually regulate the activity of the endothelial nitric oxide synthase (eNOS) in platelets. Here, we investigated the effects of insulin on the phosphorylation and activation of eNOS in washed human platelets and in endothelial cells. Insulin activated the protein kinase Akt in cultured endothelial cells and increased the phosphorylation of eNOS on Ser(1177) but failed to increase endothelial cyclic GMP levels or to elicit the relaxation of endothelium-intact porcine coronary arteries. In platelets, insulin also elicited the activation of Akt as well as the phosphorylation of eNOS and initiated NO production which was associated with increased cyclic GMP levels and the inhibition of thrombin-induced aggregation. The insulin-induced inhibition of aggregation was accompanied by a decreased Ca(2+) response to thrombin and was also prevented by N(omega) nitro-L-arginine. In platelets, but not in endothelial cells, insulin induced the activation of the AMP-activated protein kinase (AMPK), a metabolic stress-sensing kinase which was sensitive to the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the AMPK inhibitor iodotubercidin. Moreover, the insulin-mediated inhibition of thrombin-induced aggregation was prevented by iodotubercidin. Insulin-independent activation of the AMPK using 5-aminoimidazole-4-carboxamide ribonucleoside, increased platelet eNOS phosphorylation, increased cyclic GMP levels and attenuated platelet aggregation. These results highlight the differences in the signal transduction cascade activated by insulin in endothelial cells and platelets, and demonstrate that insulin stimulates the formation of NO in human platelets, in the absence of an increase in Ca(2+), by acti-vating PI3-K and AMPK which phosphorylates eNOS on Ser(1177).

  17. Hepatitis C virus core protein induces energy metabolism disorders of hepatocytes by down-regulation of silent mating type information regulation 2 homolog-1 and adenosine monophosphate-acti vated protein kinase signaling pathway

    Institute of Scientific and Technical Information of China (English)

    于建武

    2013-01-01

    Objective To study the role of silent mating type information regulation2homotog-1(SIRT1)-adenosine monophosphate(AMP)-activated protein kinase(AMPK) signaling pathway in hepatitis C virus core protein(HCV-core)induced energy metabolism disorders

  18. Coordination of Recombination with Meiotic Progression in the Caenorhabditis elegans Germline by KIN-18, a TAO Kinase That Regulates the Timing of MPK-1 Signaling.

    Science.gov (United States)

    Yin, Yizhi; Donlevy, Sean; Smolikove, Sarit

    2016-01-01

    Meiosis is a tightly regulated process requiring coordination of diverse events. A conserved ERK/MAPK-signaling cascade plays an essential role in the regulation of meiotic progression. The Thousand And One kinase (TAO) kinase is a MAPK kinase kinase, the meiotic role of which is unknown. We have analyzed the meiotic functions of KIN-18, the homolog of mammalian TAO kinases, in Caenorhabditis elegans. We found that KIN-18 is essential for normal meiotic progression; mutants exhibit accelerated meiotic recombination as detected both by analysis of recombination intermediates and by crossover outcome. In addition, ectopic germ-cell differentiation and enhanced levels of apoptosis were observed in kin-18 mutants. These defects correlate with ectopic activation of MPK-1 that includes premature, missing, and reoccurring MPK-1 activation. Late progression defects in kin-18 mutants are suppressed by inhibiting an upstream activator of MPK-1 signaling, KSR-2. However, the acceleration of recombination events observed in kin-18 mutants is largely MPK-1-independent. Our data suggest that KIN-18 coordinates meiotic progression by modulating the timing of MPK-1 activation and the progression of recombination events. The regulation of the timing of MPK-1 activation ensures the proper timing of apoptosis and is required for the formation of functional oocytes. Meiosis is a conserved process; thus, revealing that KIN-18 is a novel regulator of meiotic progression in C. elegans would help to elucidate TAO kinase's role in germline development in higher eukaryotes.

  19. Protein kinase A regulates 3-phosphatidylinositide dynamics during platelet-derived growth factor-induced membrane ruffling and chemotaxis.

    Science.gov (United States)

    Deming, Paula B; Campbell, Shirley L; Baldor, Linda C; Howe, Alan K

    2008-12-12

    Spatial regulation of the cAMP-dependent protein kinase (PKA) is required for chemotaxis in fibroblasts; however, the mechanism(s) by which PKA regulates the cell migration machinery remain largely unknown. Here we report that one function of PKA during platelet-derived growth factor (PDGF)-induced chemotaxis was to promote membrane ruffling by regulating phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) dynamics. Inhibition of PKA activity dramatically altered membrane dynamics and attenuated formation of peripheral membrane ruffles in response to PDGF. PKA inhibition also significantly decreased the number and size of PIP(3)-rich membrane ruffles in response to uniform stimulation and to gradients of PDGF. This ruffling defect was quantified using a newly developed method, based on computer vision edge-detection algorithms. PKA inhibition caused a marked attenuation in the bulk accumulation of PIP(3) following PDGF stimulation, without effects on PI3-kinase (PI3K) activity. The deficits in PIP(3) dynamics correlated with a significant inhibition of growth factor-induced membrane recruitment of endogenous Akt and Rac activation in PKA-inhibited cells. Simultaneous inhibition of PKA and Rac had an additive inhibitory effect on growth factor-induced ruffling dynamics. Conversely, the expression of a constitutively active Rac allele was able to rescue the defect in membrane ruffling and restore the localization of a fluorescent PIP(3) marker to membrane ruffles in PKA-inhibited cells, even in the absence of PI3K activity. These data demonstrate that, like Rac, PKA contributes to PIP(3) and membrane dynamics independently of direct regulation of PI3K activity and suggest that modulation of PIP(3)/3-phosphatidylinositol (3-PI) lipids represents a major target for PKA in the regulation of PDGF-induced chemotactic events.

  20. Structures of 5-Methylthioribose Kinase Reveal Substrate Specificity and Unusual Mode of Nucleotide Binding

    Energy Technology Data Exchange (ETDEWEB)

    Ku,S.; Yip, P.; Cornell, K.; Riscoe, M.; Behr, J.; Guillerm, G.; Howell, P.

    2007-01-01

    The methionine salvage pathway is ubiquitous in all organisms, but metabolic variations exist between bacteria and mammals. 5-Methylthioribose (MTR) kinase is a key enzyme in methionine salvage in bacteria and the absence of a mammalian homolog suggests that it is a good target for the design of novel antibiotics. The structures of the apo-form of Bacillus subtilis MTR kinase, as well as its ADP, ADP-PO4, AMPPCP, and AMPPCP-MTR complexes have been determined. MTR kinase has a bilobal eukaryotic protein kinase fold but exhibits a number of unique features. The protein lacks the DFG motif typically found at the beginning of the activation loop and instead coordinates magnesium via a DXE motif (Asp{sup 250}-Glu{sup 252}). In addition, the glycine-rich loop of the protein, analogous to the 'Gly triad' in protein kinases, does not interact extensively with the nucleotide. The MTR substrate-binding site consists of Asp{sup 233} of the catalytic HGD motif, a novel twin arginine motif (Arg{sup 340}/Arg{sup 341}), and a semi-conserved W-loop, which appears to regulate MTR binding specificity. No lobe closure is observed for MTR kinase upon substrate binding. This is probably because the enzyme lacks the lobe closure/inducing interactions between the C-lobe of the protein and the ribosyl moiety of the nucleotide that are typically responsible for lobe closure in protein kinases. The current structures suggest that MTR kinase has a dissociative mechanism.

  1. Drosophila salt-inducible kinase (SIK) regulates starvation resistance through cAMP-response element-binding protein (CREB)-regulated transcription coactivator (CRTC).

    Science.gov (United States)

    Choi, Sekyu; Kim, Wonho; Chung, Jongkyeong

    2011-01-28

    Salt-inducible kinase (SIK), one of the AMP-activated kinase (AMPK)-related kinases, has been suggested to play important functions in glucose homeostasis by inhibiting the cAMP-response element-binding protein (CREB)-regulated transcription coactivator (CRTC). To examine the role of SIK in vivo, we generated Drosophila SIK mutant and found that the mutant flies have higher amounts of lipid and glycogen stores and are resistant to starvation. Interestingly, SIK transcripts are highly enriched in the brain, and we found that neuron-specific expression of exogenous SIK fully rescued lipid and glycogen storage phenotypes as well as starvation resistance of the mutant. Using genetic and biochemical analyses, we demonstrated that CRTC Ser-157 phosphorylation by SIK is critical for inhibiting CRTC activity in vivo. Furthermore, double mutants of SIK and CRTC became sensitive to starvation, and the Ser-157 phosphomimetic mutation of CRTC reduced lipid and glycogen levels in the SIK mutant, suggesting that CRTC mediates the effects of SIK signaling. Collectively, our results strongly support the importance of the SIK-CRTC signaling axis that functions in the brain to maintain energy homeostasis in Drosophila.

  2. Protein kinase C (PKC) alpha and PKC theta are the major PKC isotypes involved in TCR down-regulation

    DEFF Research Database (Denmark)

    von Essen, Marina; Nielsen, Martin W; Bonefeld, Charlotte M;

    2006-01-01

    study was to identify the PKC isotype(s) involved in TCR down-regulation and to elucidate the mechanism by which they induce TCR down-regulation. To accomplish this, we studied TCR down-regulation in the human T cell line Jurkat, in primary human T cells, or in the mouse T cell line DO11.10 in which we......It is well known that protein kinase C (PKC) plays an important role in regulation of TCR cell surface expression levels. However, eight different PKC isotypes are present in T cells, and to date the particular isotype(s) involved in TCR down-regulation remains to be identified. The aim of this...... either overexpressed constitutive active or dominant-negative forms of various PKC isotypes. In addition, we studied TCR down-regulation in PKC knockout mice and by using small interfering RNA-mediated knockdown of specific PKC isotypes. We found that PKCalpha and PKCtheta were the only PKC isotypes able...

  3. Rck1 up-regulates pseudohyphal growth by activating the Ras2 and MAP kinase pathways independently in Saccharomyces cerevisiae.

    Science.gov (United States)

    Chang, Miwha; Kang, Chang-Min; Park, Yong-Sung; Yun, Cheol-Won

    2014-02-21

    Previously, we reported that Rck1 regulates Hog1 and Slt2 activities and affects MAP kinase activity in Saccharomyces cerevisiae. Recently, we found that Rck1 up-regulates phospho-Kss1 and phospho-Fus3. Kss1 has been known as a component in the pseudohyphal growth pathway, and we attempted to identify the function of Rck1 in pseudohyphal growth. Rck1 up-regulated Ras2 at the protein level, not the transcriptional level. Additionally, FLO11 transcription was up-regulated by RCK1 over-expression. RCK1 expression was up-regulated during growth on SLAD+1% butanol medium. On nitrogen starvation agar plates, RCK1 over-expression induced pseudohyphal growth of colonies, and cells over-expressing RCK1 showed a filamentous morphology when grown in SLAD medium. Furthermore, 1-butanol greatly induced filamentous growth when RCK1 was over-expressed. Moreover, invasive growth was activated in haploid cells when RCK1 was over-expressed. The growth defect of cells observed on 1-butanol medium was recovered when RCK1 was over-expressed. Interestingly, Ras2 and phospho-Kss1 were up-regulated by Rck1 independently. Together, these results suggest that Rck1 promotes pseudohyphal growth by activating Ras2 and Kss1 via independent pathways in S. cerevisiae. PMID:24491552

  4. Protein Kinase A Subunit Balance Regulates Lipid Metabolism in Caenorhabditis elegans and Mammalian Adipocytes.

    Science.gov (United States)

    Lee, Jung Hyun; Han, Ji Seul; Kong, Jinuk; Ji, Yul; Lv, Xuchao; Lee, Junho; Li, Peng; Kim, Jae Bum

    2016-09-23

    Protein kinase A (PKA) is a cyclic AMP (cAMP)-dependent protein kinase composed of catalytic and regulatory subunits and involved in various physiological phenomena, including lipid metabolism. Here we demonstrated that the stoichiometric balance between catalytic and regulatory subunits is crucial for maintaining basal PKA activity and lipid homeostasis. To uncover the potential roles of each PKA subunit, Caenorhabditis elegans was used to investigate the effects of PKA subunit deficiency. In worms, suppression of PKA via RNAi resulted in severe phenotypes, including shortened life span, decreased egg laying, reduced locomotion, and altered lipid distribution. Similarly, in mammalian adipocytes, suppression of PKA regulatory subunits RIα and RIIβ via siRNAs potently stimulated PKA activity, leading to potentiated lipolysis without increasing cAMP levels. Nevertheless, insulin exerted anti-lipolytic effects and restored lipid droplet integrity by antagonizing PKA action. Together, these data implicate the importance of subunit stoichiometry as another regulatory mechanism of PKA activity and lipid metabolism.

  5. The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover

    DEFF Research Database (Denmark)

    Monaghan, Jacqueline; Matschi, Susanne; Shorinola, Oluwaseyi;

    2014-01-01

    Plant perception of pathogen-associated molecular patterns (PAMPs) triggers a phosphorylation relay leading to PAMP-triggered immunity (PTI). Despite increasing knowledge of PTI signaling, how immune homeostasis is maintained remains largely unknown. Here we describe a forward-genetic screen to i...... contributes to BIK1 turnover. Our results suggest a negative regulatory mechanism that continually buffers immune signaling by controlling the turnover of this key signaling kinase....

  6. Polo kinase Cdc5 is a central regulator of meiosis I

    OpenAIRE

    Attner, Michelle A.; Miller, Matthew P.; Ee, Ly-Sha; Elkin, Sheryl K; Amon, Angelika

    2013-01-01

    During meiosis, two consecutive rounds of chromosome segregation yield four haploid gametes from one diploid cell. The Polo kinase Cdc5 is required for meiotic progression, but how Cdc5 coordinates multiple cell-cycle events during meiosis I is not understood. Here we show that CDC5-dependent phosphorylation of Rec8, a subunit of the cohesin complex that links sister chromatids, is required for efficient cohesin removal from chromosome arms, which is a prerequisite for meiosis I chromosome se...

  7. Regulation of pyruvate dehydrogenase kinase activity by protein thiol-disulfide exchange.

    OpenAIRE

    Pettit, F H; Humphreys, J; Reed, L J

    1982-01-01

    Endogenous kinase activity of highly purified pyruvate dehydrogenase complex from bovine kidney is markedly inhibited by N-ethylmaleimide and by certain disulfides. Inhibition by disulfides is highly specific and is reversed by thiols. 5,5'-Dithiobis(2-nitrobenzoate) is the most potent inhibitor, showing significant inhibition at a concentration as low as 1 microM. Cystamine, oxidized glutathione, pantethine, lipoic acid, lipoamide, ergothionine, insulin, oxytocin, and vasopressin were ineffe...

  8. Mechanisms of regulation and function of G-protein-coupled receptor kinases

    Institute of Scientific and Technical Information of China (English)

    Wen Yang; Shi-Hai Xia

    2006-01-01

    G-protein-coupled receptor kinases (GRKs) interact with the agonist-activated form of G-protein-coupled receptor (GPCR) to affect receptor phosphorylation and to initiate profound impairment of receptor signaling,or desensitization. GPCR forms the largest family of cell surface receptors, and defects in GRK function have the potential consequence to affect GPCR-stimulated biological responses in many pathological situations.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  10. A translational regulator, PUM2, promotes both protein stability and kinase activity of Aurora-A.

    Directory of Open Access Journals (Sweden)

    Yei-Hsuan Huang

    Full Text Available Aurora-A, a centrosomal serine-threonine kinase, orchestrates several key aspects of cell division. However, the regulatory pathways for the protein stability and kinase activity of Aurora-A are still not completely understood. In this study, PUM2, an RNA-binding protein, is identified as a novel substrate and interacting protein of Aurora-A. Overexpression of the PUM2 mutant which fails to interact with Aurora-A, and depletion of PUM2 result in a decrease in the amount of Aurora-A. PUM2 physically binds to the D-box of Aurora-A, which is recognized by APC/C(Cdh1. Overexpression of PUM2 prevents ubiquitination and enhances the protein stability of Aurora-A, suggesting that PUM2 protects Aurora-A from APC/C(Cdh1-mediated degradation. Moreover, association of PUM2 with Aurora-A not only makes Aurora-A more stable but also enhances the kinase activity of Aurora-A. Our study suggests that PUM2 plays two different but important roles during cell cycle progression. In interphase, PUM2 localizes in cytoplasm and plays as translational repressor through its RNA binding domain. However, in mitosis, PUM2 physically associates with Aurora-A to ensure enough active Aurora-A at centrosomes for mitotic entry. This is the first time to reveal the moonlight role of PUM2 in mitosis.

  11. Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases.

    Science.gov (United States)

    Steger, Martin; Tonelli, Francesca; Ito, Genta; Davies, Paul; Trost, Matthias; Vetter, Melanie; Wachter, Stefanie; Lorentzen, Esben; Duddy, Graham; Wilson, Stephen; Baptista, Marco As; Fiske, Brian K; Fell, Matthew J; Morrow, John A; Reith, Alastair D; Alessi, Dario R; Mann, Matthias

    2016-01-01

    Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD. PMID:26824392

  12. α1A-adrenergic receptor induces activation of extracellular signal-regulated kinase 1/2 through endocytic pathway.

    Directory of Open Access Journals (Sweden)

    Fei Liu

    Full Text Available G protein-coupled receptors (GPCRs activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α(1A-adrenergic receptor (α(1A-AR-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2. Agonist-mediated endocytic traffic of α(1A-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A. α(1A-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α(1A-AR. α(1A-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent. Activation of protein kinase C (PKC and C-Raf by α(1A-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor and Ro 31-8220 (a PKC inhibitor inhibited α(1B-AR- but not α(1A-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α(1A-AR-induced ERK1/2 activation, which is independent of G(q/PLC/PKC signaling.

  13. 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...

  14. 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...

  15. Enhanced transcription of contractile 5-hydroxytryptamine 2A receptors via extracellular signal-regulated kinase 1/2 after organ culture of rat mesenteric artery

    DEFF Research Database (Denmark)

    Cao, Yong-Xiao; He, Lang-Chong; Xu, Cang-Bao;

    2005-01-01

    5-Hydroxytryptamine (5-HT) has been found to elicit enhanced contractile effects in some vascular disorders. The present study was designed to examine if vascular 5-HT2A receptors are up-regulated during organ culture and if the extracellular signal-regulated protein kinase 1/2 (ERK1/2) pathways ...

  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. Inhibition of host extracellular signal-regulated kinase (ERK) activation decreases new world alphavirus multiplication in infected cells

    Energy Technology Data Exchange (ETDEWEB)

    Voss, Kelsey; Amaya, Moushimi [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States); Mueller, Claudius [Center for Applied Proteomics and Personalized Medicine, George Mason University, 10900 University Boulevard, Manassas, VA (United States); Roberts, Brian [Leidos Health Life Sciences, 5202 Presidents Court, Suite 110, Frederick, MD (United States); Kehn-Hall, Kylene; Bailey, Charles [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States); Petricoin, Emanuel [Center for Applied Proteomics and Personalized Medicine, George Mason University, 10900 University Boulevard, Manassas, VA (United States); Narayanan, Aarthi, E-mail: anaraya1@gmu.edu [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States)

    2014-11-15

    New World alphaviruses belonging to the family Togaviridae are classified as emerging infectious agents and Category B select agents. Our study is focused on the role of the host extracellular signal-regulated kinase (ERK) in the infectious process of New World alphaviruses. Infection of human cells by Venezuelan equine encephalitis virus (VEEV) results in the activation of the ERK-signaling cascade. Inhibition of ERK1/2 by the small molecule inhibitor Ag-126 results in inhibition of viral multiplication. Ag-126-mediated inhibition of VEEV was due to potential effects on early and late stages of the infectious process. While expression of viral proteins was down-regulated in Ag-126 treated cells, we did not observe any influence of Ag-126 on the nuclear distribution of capsid. Finally, Ag-126 exerted a broad-spectrum inhibitory effect on New World alphavirus multiplication, thus indicating that the host kinase, ERK, is a broad-spectrum candidate for development of novel therapeutics against New World alphaviruses. - Highlights: • VEEV infection activated multiple components of the ERK signaling cascade. • Inhibition of ERK activation using Ag-126 inhibited VEEV multiplication. • Activation of ERK by Ceramide C6 increased infectious titers of TC-83. • Ag-126 inhibited virulent strains of all New World alphaviruses. • Ag-126 treatment increased percent survival of infected cells.

  19. Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO

    Energy Technology Data Exchange (ETDEWEB)

    Reidick, Christina [Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum 44801 (Germany); El Magraoui, Fouzi; Meyer, Helmut E. [Biomedical Research, Human Brain Proteomics II, Leibniz-Institut für Analytische Wissenschaften-ISAS, Dortmund 44139 (Germany); Stenmark, Harald [Department of Biochemistry, Institute for Cancer Research, Oslo University Hospital, Montebello, Oslo 0310 (Norway); Platta, Harald W., E-mail: harald.platta@rub.de [Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum 44801 (Germany)

    2014-12-23

    The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept.

  20. NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

    Science.gov (United States)

    Kuek, Vincent; Yang, Zhifan; Chim, Shek Man; Zhu, Sipin; Xu, Huazi; Chow, Siu To; Tickner, Jennifer; Rosen, Vicki; Erber, Wendy; Li, Xiucheng; An, Qin; Qian, Yu; Xu, Jiake

    2016-01-01

    Angiogenesis plays an important role in bone development and remodeling and is mediated by a plethora of potential angiogenic factors. However, data regarding specific angiogenic factors that are secreted within the bone microenvironment to regulate osteoporosis is lacking. Here, we report that Nephronectin (NPNT), a member of the epidermal growth factor (EGF) repeat superfamily proteins and a homologue of EGFL6, is expressed in osteoblasts. Intriguingly, the gene expression of NPNT is reduced in the bone of C57BL/6J ovariectomised mice and in osteoporosis patients. In addition, the protein levels of NPNT and CD31 are also found to be reduced in the tibias of OVX mice. Exogenous addition of mouse recombinant NPNT on endothelial cells stimulates migration and tube-like structure formation in vitro. Furthermore, NPNT promotes angiogenesis in an ex vivo fetal mouse metatarsal angiogenesis assay. We show that NPNT stimulates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated kinase (MAPK) in endothelial cells. Inhibition of ERK1/2 impaired NPNT-induced endothelial cell migration, tube-like structure formation and angiogenesis. Taken together, these results demonstrate that NPNT is a paracrine angiogenic factor and may play a role in pathological osteoporosis. This may lead to new targets for treatment of bone diseases and injuries. PMID:27782206

  1. Lemur tyrosine kinase-2 signalling regulates kinesin-1 light chain-2 phosphorylation and binding of Smad2 cargo.

    LENUS (Irish Health Repository)

    Manser, C

    2012-05-31

    A recent genome-wide association study identified the gene encoding lemur tyrosine kinase-2 (LMTK2) as a susceptibility gene for prostate cancer. The identified genetic alteration is within intron 9, but the mechanisms by which LMTK2 may impact upon prostate cancer are not clear because the functions of LMTK2 are poorly understood. Here, we show that LMTK2 regulates a known pathway that controls phosphorylation of kinesin-1 light chain-2 (KLC2) by glycogen synthase kinase-3β (GSK3β). KLC2 phosphorylation by GSK3β induces the release of cargo from KLC2. LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3β on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2. Smad2 signals to the nucleus in response to transforming growth factor-β (TGFβ) receptor stimulation and transport of Smad2 by kinesin-1 is required for this signalling. We show that small interfering RNA loss of LMTK2 not only reduces binding of Smad2 to KLC2, but also inhibits TGFβ-induced Smad2 signalling. Thus, LMTK2 may regulate the activity of kinesin-1 motor function and Smad2 signalling.

  2. The mammalian AMP-activated protein kinase complex mediates glucose regulation of gene expression in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Ye, Tian; Bendrioua, Loubna; Carmena, David; García-Salcedo, Raúl; Dahl, Peter; Carling, David; Hohmann, Stefan

    2014-06-01

    The AMP-activated protein kinase (AMPK) controls energy homeostasis in eukaryotic cells. Here we expressed hetero-trimeric mammalian AMPK complexes in a Saccharomyces cerevisiae mutant lacking all five genes encoding yeast AMPK/SNF1 components. Certain mammalian complexes complemented the growth defect of the yeast mutant on non-fermentable carbon sources. Phosphorylation of the AMPK α1-subunit was glucose-regulated, albeit not by the Glc7-Reg1/2 phosphatase, which performs this function on yeast AMPK/SNF1. AMPK could take over SNF1 function in glucose derepression. While indirectly acting anti-diabetic drugs had no effect on AMPK in yeast, compound 991 stimulated α1-subunit phosphorylation. Our results demonstrate a remarkable functional conservation of AMPK and that glucose regulation of AMPK may not be mediated by regulatory features of a specific phosphatase.

  3. Up-regulation of endothelin type B receptors in the human internal mammary artery in culture is dependent on protein kinase C and mitogen-activated kinase signaling pathways

    DEFF Research Database (Denmark)

    Nilsson, David; Gustafsson, Lotta; Wackenfors, Angelica;

    2008-01-01

    Up-regulation of vascular endothelin type B (ETB) receptors is implicated in the pathogenesis of cardiovascular disease. Culture of intact arteries has been shown to induce similar receptor alterations and has therefore been suggested as a suitable method for, ex vivo, in detail delineation of th...... of the regulation of endothelin receptors. We hypothesize that mitogen-activated kinases (MAPK) and protein kinase C (PKC) are involved in the regulation of endothelin ETB receptors in human internal mammary arteries.......Up-regulation of vascular endothelin type B (ETB) receptors is implicated in the pathogenesis of cardiovascular disease. Culture of intact arteries has been shown to induce similar receptor alterations and has therefore been suggested as a suitable method for, ex vivo, in detail delineation...

  4. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

    Science.gov (United States)

    Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

    2015-05-29

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.

  5. LRRK2 Kinase Activity and Biology are Not Uniformly Predicted by its Autophosphorylation and Cellular Phosphorylation Site Status

    Directory of Open Access Journals (Sweden)

    April eReynolds

    2014-06-01

    Full Text Available Missense mutations in the Leucine Rich Repeat protein Kinase 2 (LRRK2 gene are the most common genetic predisposition to develop Parkinson’s disease (PD LRRK2 is a large multi-domain phosphoprotein with a GTPase domain and a serine/threonine protein kinase domain whose activity is implicated in neuronal toxicity; however the precise mechanism is unknown. LRRK2 autophosphorylates on several serine/threonine residues across the enzyme and is found constitutively phosphorylated on Ser910, Ser935, Ser955 and Ser973, which are proposed to be regulated by upstream kinases. Here we investigate the phosphoregulation at these sites by analyzing the effects of disease-associated mutations Arg1441Cys, Arg1441Gly, Ala1442Pro, Tyr1699Cys, Ile2012Thr, Gly2019Ser, and Ile2020Thr. We also studied alanine substitutions of phosphosite serines 910, 935, 955 and 973 and specific LRRK2 inhibition on autophosphorylation of LRRK2 Ser1292, Thr1491, Thr2483 and phosphorylation at the cellular sites. We found that mutants in the Roc-COR domains, including Arg1441Cys, Arg1441His, Ala1442Pro and Tyr1699Cys, can positively enhance LRRK2 kinase activity while concomitantly inducing the dephosphorylation of the cellular sites. Mutation of the cellular sites individually did not affect LRRK2 intrinsic kinase activity; however, Ser910/935/955/973Ala mutations trended toward increased kinase activity of LRRK2. Increased cAMP levels did not lead to increased LRRK2 cellular site phosphorylation, 14-3-3 binding or kinase activity. In cells, inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser1292 by Calyculin A and okadaic acid sensitive phosphatases, while the cellular sites are dephosphorylated by Calyculin A sensitive phosphatases. These findings indicate that comparative analysis of both Ser1292 and Ser910/935/955/973 phosphorylation sites will provide important and distinct measures of LRRK2 kinase and biological activity in vitro and in vivo.

  6. Arabidopsis MAP kinase 4 regulates salicylic acid- and jasmonic acid/ethylene-dependent responses via EDS1 and PAD4.

    Science.gov (United States)

    Brodersen, Peter; Petersen, Morten; Bjørn Nielsen, Henrik; Zhu, Shijiang; Newman, Mari-Anne; Shokat, Kevan M; Rietz, Steffen; Parker, Jane; Mundy, John

    2006-08-01

    Arabidopsis MPK4 has been implicated in plant defense regulation because mpk4 knockout plants exhibit constitutive activation of salicylic acid (SA)-dependent defenses, but fail to induce jasmonic acid (JA) defense marker genes in response to JA. We show here that mpk4 mutants are also defective in defense gene induction in response to ethylene (ET), and that they are more susceptible than wild-type (WT) to Alternaria brassicicola that induces the ET/JA defense pathway(s). Both SA-repressing and ET/JA-(co)activating functions depend on MPK4 kinase activity and involve the defense regulators EDS1 and PAD4, as mutations in these genes suppress de-repression of the SA pathway and suppress the block of the ET/JA pathway in mpk4. EDS1/PAD4 thus affect SA-ET/JA signal antagonism as activators of SA but as repressors of ET/JA defenses, and MPK4 negatively regulates both of these functions. We also show that the MPK4-EDS1/PAD4 branch of ET defense signaling is independent of the ERF1 transcription factor, and use comparative microarray analysis of ctr1, ctr1/mpk4, mpk4 and WT to show that MPK4 is required for induction of a small subset of ET-regulated genes. The regulation of some, but not all, of these genes involves EDS1 and PAD4.

  7. DNA damage checkpoint kinase ATM regulates germination and maintains genome stability in seeds.

    Science.gov (United States)

    Waterworth, Wanda M; Footitt, Steven; Bray, Clifford M; Finch-Savage, William E; West, Christopher E

    2016-08-23

    Genome integrity is crucial for cellular survival and the faithful transmission of genetic information. The eukaryotic cellular response to DNA damage is orchestrated by the DNA damage checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR). Here we identify important physiological roles for these sensor kinases in control of seed germination. We demonstrate that double-strand breaks (DSBs) are rate-limiting for germination. We identify that desiccation tolerant seeds exhibit a striking transcriptional DSB damage response during germination, indicative of high levels of genotoxic stress, which is induced following maturation drying and quiescence. Mutant atr and atm seeds are highly resistant to aging, establishing ATM and ATR as determinants of seed viability. In response to aging, ATM delays germination, whereas atm mutant seeds germinate with extensive chromosomal abnormalities. This identifies ATM as a major factor that controls germination in aged seeds, integrating progression through germination with surveillance of genome integrity. Mechanistically, ATM functions through control of DNA replication in imbibing seeds. ATM signaling is mediated by transcriptional control of the cell cycle inhibitor SIAMESE-RELATED 5, an essential factor required for the aging-induced delay to germination. In the soil seed bank, seeds exhibit increased transcript levels of ATM and ATR, with changes in dormancy and germination potential modulated by environmental signals, including temperature and soil moisture. Collectively, our findings reveal physiological functions for these sensor kinases in linking genome integrity to germination, thereby influencing seed quality, crucial for plant survival in the natural environment and sustainable crop production. PMID:27503884

  8. Protocadherin clusters and cell adhesion kinase regulate dendrite complexity through Rho GTPase

    Institute of Scientific and Technical Information of China (English)

    Lun Suo; Huinan Lu; Guoxin Ying; Mario R. Capecchi; Qiang Wu

    2012-01-01

    Dendritic patterning and spine morphogenesis are crucial for the assembly of neuronal circuitry to ensure normal brain development and synaptic connectivity as well as for understanding underlying mechanisms of neuropsychiatric diseases and cognitive impairments.The Rho GTPase family is essential for neuronal morphogenesis and synaptic plasticity by modulating and reorganizing the cytoskeleton.Here,we report that protocadherin (Pcdh) clusters and cell adhesion kinases (CAKs) play important roles in dendritic development and spine elaboration.The knockout of the entire Pcdhα cluster results in the dendritic simplification and spine loss in CA1 pyramidal neurons in vivo and in cultured primary hippocampal neurons in vitro.The knockdown of the whole Pcdhγ cluster or in combination with the Pcdhα knockout results in similar dendritic and spine defects in vitro.The overexpression of proline-rich tyrosine kinase 2 (Pyk2,also known as CAKβ,RAFTK,FAK2,and CADTK) recapitulates these defects and its knockdown rescues the phenotype.Moreover,the genetic deletion of the Pcdhα cluster results in phosphorylation and activation of Pyk2 and focal adhesion kinase (Fak) and the inhibition of Rho GTPases in vivo.Finally,the overexpression of PyK2 leads to inactivation of Rac1 and,conversely,the constitutive active Rac1 rescues the dendritic and spine morphogenesis defects caused by the knockout of the Pcdhα cluster and the knockdown of the Pcdhγ cluster.Thus,the involvement of the Pcdh-CAK-Rho GTPase pathway in the dendritic development and spine morphogenesis has interesting implications for proper assembly of neuronal connections in the brain.

  9. The insulin receptor C-terminus is involved in regulation of the receptor kinase activity.

    Science.gov (United States)

    Kaliman, P; Baron, V; Alengrin, F; Takata, Y; Webster, N J; Olefsky, J M; Van Obberghen, E

    1993-09-21

    During the insulin receptor activation process, ligand binding and autophosphorylation induce two distinct conformational changes in the C-terminal domain of the receptor beta-subunit. To analyze the role of this domain and the involvement of the C-terminal autophosphorylation sites (Tyr1316 and Tyr1322) in receptor activation, we used (i) antipeptide antibodies against three different C-terminal sequences (1270-1281, 1294-1317, and 1309-1326) and (ii) an insulin receptor mutant (Y/F2) where Tyr1316 and Tyr1322 have been replaced by Phe. We show that the autophosphorylation-induced C-terminal conformational change is preserved in the Y/F2 receptor, indicating that this change is not induced by phosphorylation of the C-terminal sites but most likely by phosphorylation of the major sites in the kinase domain (Tyr1146, Tyr1150, and Tyr1151). Binding of antipeptide antibodies to the C-terminal domain modulated (activated or inhibited) both mutant and wild-type receptor-mediated phosphorylation of poly(Glu/Tyr). In contrast to the wild-type receptor, Y/F2 exhibited the same C-terminal configuration before and after insulin binding, evidencing that mutation of Tyr1316 and Tyr1322 introduced conformational changes in the C-terminus. Finally, the mutant receptor was 2-fold more active than the wild-type receptor for poly(Glu/Tyr) phosphorylation. In conclusion, the whole C-terminal region of the insulin receptor beta-subunit is likely to exert a regulatory influence on the receptor kinase activity. Perturbations of the C-terminal region, such as binding of antipeptides or mutation of Tyr1316 and Tyr1322, provoke alterations at the receptor kinase level, leading to activation or inhibition of the enzymic activity. PMID:7690586

  10. Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

    Directory of Open Access Journals (Sweden)

    Colpo Anna

    2010-10-01

    Full Text Available Abstract Background Glycogen Synthase Kinase-3 (GSK-3 α and β are two serine-threonine kinases controlling insulin, Wnt/β-catenin, NF-κB signaling and other cancer-associated transduction pathways. Recent evidence suggests that GSK-3 could function as growth-promoting kinases, especially in malignant cells. In this study, we have investigated GSK-3α and GSK-3β function in multiple myeloma (MM. Methods GSK-3 α and β expression and cellular localization were investigated by Western blot (WB and immunofluorescence analysis in a panel of MM cell lines and in freshly isolated plasma cells from patients. MM cell growth, viability and sensitivity to bortezomib was assessed upon treatment with GSK-3 specific inhibitors or transfection with siRNAs against GSK-3 α and β isoforms. Survival signaling pathways were studied with WB analysis. Results GSK-3α and GSK-3β were differently expressed and phosphorylated in MM cells. Inhibition of GSK-3 with the ATP-competitive, small chemical compounds SB216763 and SB415286 caused MM cell growth arrest and apoptosis through the activation of the intrinsic pathway. Importantly, the two inhibitors augmented the bortezomib-induced MM cell cytotoxicity. RNA interference experiments showed that the two GSK-3 isoforms have distinct roles: GSK-3β knock down decreased MM cell viability, while GSK-3α knock down was associated with a higher rate of bortezomib-induced cytotoxicity. GSK-3 inhibition caused accumulation of β-catenin and nuclear phospho-ERK1, 2. Moreover, GSK-3 inhibition and GSK-3α knockdown enhanced bortezomib-induced AKT and MCL-1 protein degradation. Interestingly, bortezomib caused a reduction of GSK-3 serine phosphorylation and its nuclear accumulation with a mechanism that resulted partly dependent on GSK-3 itself. Conclusions These data suggest that in MM cells GSK-3α and β i play distinct roles in cell survival and ii modulate the sensitivity to proteasome inhibitors.

  11. DNA damage checkpoint kinase ATM regulates germination and maintains genome stability in seeds.

    Science.gov (United States)

    Waterworth, Wanda M; Footitt, Steven; Bray, Clifford M; Finch-Savage, William E; West, Christopher E

    2016-08-23

    Genome integrity is crucial for cellular survival and the faithful transmission of genetic information. The eukaryotic cellular response to DNA damage is orchestrated by the DNA damage checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR). Here we identify important physiological roles for these sensor kinases in control of seed germination. We demonstrate that double-strand breaks (DSBs) are rate-limiting for germination. We identify that desiccation tolerant seeds exhibit a striking transcriptional DSB damage response during germination, indicative of high levels of genotoxic stress, which is induced following maturation drying and quiescence. Mutant atr and atm seeds are highly resistant to aging, establishing ATM and ATR as determinants of seed viability. In response to aging, ATM delays germination, whereas atm mutant seeds germinate with extensive chromosomal abnormalities. This identifies ATM as a major factor that controls germination in aged seeds, integrating progression through germination with surveillance of genome integrity. Mechanistically, ATM functions through control of DNA replication in imbibing seeds. ATM signaling is mediated by transcriptional control of the cell cycle inhibitor SIAMESE-RELATED 5, an essential factor required for the aging-induced delay to germination. In the soil seed bank, seeds exhibit increased transcript levels of ATM and ATR, with changes in dormancy and germination potential modulated by environmental signals, including temperature and soil moisture. Collectively, our findings reveal physiological functions for these sensor kinases in linking genome integrity to germination, thereby influencing seed quality, crucial for plant survival in the natural environment and sustainable crop production.

  12. Constitutive hypophosphorylation of extracellular signal-regulated kinases-1/2 and down-regulation of c-Jun in human gastric adenocarcinoma

    International Nuclear Information System (INIS)

    Hyperphosphorylation of extracellular signal-regulated protein kinases-1/2 (ERK1/2) is known to promote cancer cell proliferation. We therefore investigated the constitutive phosphorylation levels of ERK1/2 and the expression of its downstream targets c-Fos, c-Jun, and cyclooxygenase-2 (COX-2) in biopsied human gastric cancer tissues. Results showed that ERK1/2 phosphorylation and c-Jun expression were significantly lowered in gastric cancer compared with the non-cancer adjacent tissues. The expression of c-Fos, however, was not altered while COX-2 was significantly up-regulated. To conclude, we demonstrate that hypophosphorylation of ERK1/2 may occur in gastric cancer. Such discovery may have implication in the application of pathway-directed therapy for this malignant disease

  13. Regulation of the Drosophila melanogaster Protein, Enhancer of Rudimentary, by Casein Kinase II

    OpenAIRE

    Gelsthorpe, Mark E.; Tan, Zehui; Phillips, Anthony; Eissenberg, Joel C.; Miller, Ashley; Wallace, Janell; Tsubota, Stuart I.

    2006-01-01

    The Drosophila melanogaster gene enhancer of rudimentary, e(r), encodes a conserved protein, ER. Most ER homologs share two casein kinase II (CKII) target sites. In D. melanogaster, these sites are T18 and S24. A third CKII site, T63, has been seen only in drosophilids. The conservation of these CKII sites, particularly T18 and S24, suggests a role for these residues in the function of the protein. To test this hypothesis, these positions were mutated either to alanine as a nonphosphorylated ...

  14. Regulation of the RNA-dependent protein kinase by triple helix formation

    OpenAIRE

    Vuyisich, Momchilo; Beal, Peter A.

    2000-01-01

    The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates the α-subunit of the translation initiation factor eIF-2, inhibiting its function. PKR is activated in vitro by binding to double-stranded RNA (dsRNA) molecules of ~30 bp or longer. Here we show that triple helix forming oligonucleotides (TFOs) inhibit dsRNA binding to the isolated RNA binding domain of PKR. The inhibition is specific to the targeted RNA and dependent on TFO length. Bindi...

  15. Reduced Mechanical Stretch Induces Enhanced Endothelin B Receptor-mediated Contractility via Activation of Focal Adhesion Kinase and Extra Cellular-regulated Kinase 1/2 in Cerebral Arteries from Rat

    DEFF Research Database (Denmark)

    Rasmussen, Marianne N P; Spray, Stine; Skovsted, Gry F;

    2016-01-01

    Cerebral ischaemia results in enhanced endothelin B (ETB ) receptor-mediated contraction and receptor protein expression in the affected cerebrovascular smooth muscle cells (SMC). Organ culture of cerebral arteries is a method to induce similar alterations in ETB receptor expression. We hypothesize...... ETB receptor agonist sarafotoxin 6c. The involvement of extracellular regulated kinase (ERK) 1/2 and focal adhesion kinase (FAK) were studied by their specific inhibitors U0126 and PF-228, respectively. Compared to their stretched counterparts, un-stretched MCA segments showed a significantly...

  16. 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.

  17. Mechanisms of extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signaltransduction pathway in depressive disorder

    Institute of Scientific and Technical Information of China (English)

    Hongyan Wang; Yingquan Zhang; Mingqi Qiao

    2013-01-01

    The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway plays an important role in the mechanism of action of antidepressant drugs and has dominated recent studies on the pathogenesis of depression. In the present review we summarize the known roles of extracellular signal-regulated kinase, cAMP response element-binding protein and brain-derived neurotrophic factor in the pathogenesis of depression and in the mechanism of action of antidepressant medicines. The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway has potential to be used as a biological index to help diagnose depression, and as such it is considered as an important new target in the treatment of depression.

  18. Spleen Tyrosine Kinase Regulates AP-1 Dependent Transcriptional Response to Minimally Oxidized LDL

    Science.gov (United States)

    Choi, Soo-Ho; Wiesner, Philipp; Almazan, Felicidad; Kim, Jungsu; Miller, Yury I.

    2012-01-01

    Oxidative modification of low-density lipoprotein (LDL) turns it into an endogenous ligand recognized by pattern-recognition receptors. We have demonstrated that minimally oxidized LDL (mmLDL) binds to CD14 and mediates TLR4/MD-2-dependent responses in macrophages, many of which are MyD88-independent. We have also demonstrated that the mmLDL activation leads to recruitment of spleen tyrosine kinase (Syk) to TLR4 and TLR4 and Syk phosphorylation. In this study, we produced a macrophage-specific Syk knockout mouse and used primary Syk−/− macrophages in our studies. We demonstrated that Syk mediated phosphorylation of ERK1/2 and JNK, which in turn phosphorylated c-Fos and c-Jun, respectively, as assessed by an in vitro kinase assay. c-Jun phosphorylation was also mediated by IKKε. c-Jun and c-Fos bound to consensus DNA sites and thereby completed an AP-1 transcriptional complex and induced expression of CXCL2 and IL-6. These results suggest that Syk plays a key role in TLR4-mediated macrophage responses to host-generated ligands, like mmLDL, with subsequent activation of an AP-1 transcription program. PMID:22384232

  19. TOUSLED Kinase Activity Oscillates during the Cell Cycle and Interacts with Chromatin Regulators1

    Science.gov (United States)

    Ehsan, Hashimul; Reichheld, Jean-Philippe; Durfee, Tim; Roe, Judith L.

    2004-01-01

    The TOUSLED (TSL)-like nuclear protein kinase family is highly conserved in plants and animals. tsl loss of function mutations cause pleiotropic defects in both leaf and flower development, and growth and initiation of floral organ primordia is abnormal, suggesting that basic cellular processes are affected. TSL is more highly expressed in exponentially growing Arabidopsis culture cells than in stationary, nondividing cells. While its expression remains constant throughout the cell cycle in dividing cells, TSL kinase activity is higher in enriched late G2/M-phase and G1-phase populations of Arabidopsis suspension culture cells compared to those in S-phase. tsl mutants also display an aberrant pattern and increased expression levels of the mitotic cyclin gene CycB1;1, suggesting that TSL represses CycB1;1 expression at certain times during development or that cells are delayed in mitosis. TSL interacts with and phosphorylates one of two Arabidopsis homologs of the nucleosome assembly/silencing protein Asf1 and histone H3, as in humans, and a novel plant SANT/myb-domain protein, TKI1, suggesting that TSL plays a role in chromatin metabolism. PMID:15047893

  20. Myotonic dystrophy protein kinase phosphorylates phospholamban and regulates calcium uptake in cardiomyocyte sarcoplasmic reticulum.

    Science.gov (United States)

    Kaliman, Perla; Catalucci, Daniele; Lam, Jason T; Kondo, Richard; Gutiérrez, José Carlos Paz; Reddy, Sita; Palacín, Manuel; Zorzano, Antonio; Chien, Kenneth R; Ruiz-Lozano, Pilar

    2005-03-01

    Myotonic dystrophy (DM) is caused by a CTG expansion in the 3'-untranslated region of a protein kinase gene (DMPK). Cardiovascular disease is one of the most prevalent causes of death in DM patients. Electrophysiological studies in cardiac muscles from DM patients and from DMPK(-/-) mice suggested that DMPK is critical to the modulation of cardiac contractility and to the maintenance of proper cardiac conduction activity. However, there are no data regarding the molecular signaling pathways involved in DM heart failure. Here we show that DMPK expression in cardiac myocytes is highly enriched in the sarcoplasmic reticulum (SR) where it colocalizes with the ryanodine receptor and phospholamban (PLN), a muscle-specific SR Ca(2+)-ATPase (SERCA2a) inhibitor. Coimmunoprecipitation studies showed that DMPK and PLN can physically associate. Furthermore, purified wild-type DMPK, but not a kinase-deficient mutant (K110A DMPK), phosphorylates PLN in vitro. Subsequent studies using the DMPK(-/-) mice demonstrated that PLN is hypo-phosphorylated in SR vesicles from DMPK(-/-) mice compared with wild-type mice both in vitro and in vivo. Finally, we show that Ca(2+) uptake in SR is impaired in ventricular homogenates from DMPK(-/-) mice. Together, our data suggest the existence of a novel regulatory DMPK pathway for cardiac contractility and provide a molecular mechanism for DM heart pathology. PMID:15598648

  1. Regulation of cell signaling dynamics by the protein kinase-scaffold Ste5.

    Science.gov (United States)

    Hao, Nan; Nayak, Sujata; Behar, Marcelo; Shanks, Ryan H; Nagiec, Michal J; Errede, Beverly; Hasty, Jeffrey; Elston, Timothy C; Dohlman, Henrik G

    2008-06-01

    Cell differentiation requires the ability to detect and respond appropriately to a variety of extracellular signals. Here we investigate a differentiation switch induced by changes in the concentration of a single stimulus. Yeast cells exposed to high doses of mating pheromone undergo cell division arrest. Cells at intermediate doses become elongated and divide in the direction of a pheromone gradient (chemotropic growth). Either of the pheromone-responsive MAP kinases, Fus3 and Kss1, promotes cell elongation, but only Fus3 promotes chemotropic growth. Whereas Kss1 is activated rapidly and with a graded dose-response profile, Fus3 is activated slowly and exhibits a steeper dose-response relationship (ultrasensitivity). Fus3 activity requires the scaffold protein Ste5; when binding to Ste5 is abrogated, Fus3 behaves like Kss1, and the cells no longer respond to a gradient or mate efficiently with distant partners. We propose that scaffold proteins serve to modulate the temporal and dose-response behavior of the MAP kinase. PMID:18538663

  2. Nemo like kinase negatively regulates NF-κB activation and coelomocytes apoptosis in Apostichopus japonicus.

    Science.gov (United States)

    Lv, Zhimeng; Li, Chenghua; Zhang, Weiwei; Jin, Chunua; Shao, Yina; Xuemei, Duan; Qingxi, Han

    2016-01-01

    Nuclear factor kappa B (NF-κB) transcription factors are related to several physiological processes, including innate and acquired immunity. In this study, a novel negative regulator of the Nemo-like kinase (NLK) gene was identified from Apostichopus japonicus through PCR (denoted as AjNLK). The complete AjNLK cDNA was of 2335 bp, with a 5'-UTR of 315 bp, a 3'-UTR of 718 bp, and a putative ORF of 1302 bp, and encoded a polypeptide of 433 amino acid residues with a typical serine/threonine protein kinase domain. Blast analysis revealed that AjNLK shared a high degree of structural conservation with its counterparts from other invertebrates and vertebrates. Spatial expression analysis indicated that the expression of AjNLK mRNA transcripts was higher in the tentacles than that in coelomocytes. The expression of AjNLK mRNA in coelomocytes was suppressed after Vibrio splendidus challenge by 0.51-fold and 0.41-fold at 72 and 96 h, respectively, compared with that in the control group. Similarly, AjNLK expression was down-regulated in primary coelomocytes exposed to 1 μg mL(-1) lipopolysaccharide (LPS). Functional investigation further revealed that the NF-κB factor p105 was induced at both mRNA and protein levels after AjNLK silencing in vitro. Meanwhile, the apoptosis of LPS-induced coelomocytes was significantly inhibited in AjNLK siRNA-transfected coelomocytes. These results supported that AjNLK negatively regulated NF-κB activation and cell apoptosis in sea cucumber. PMID:26363086

  3. Protein Kinase C-δ mediates down-regulation of heterogeneous nuclear ribonucleoprotein K protein: involvement in apoptosis induction

    International Nuclear Information System (INIS)

    We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase C delta (ΔPKC-δ). By subcellular proteome analysis, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was identified as being significantly down-regulated in NSC606985-treated leukemic NB4 cells. HnRNP K, a docking protein for DNA, RNA, and transcriptional or translational molecules, is implicated in a host of processes involving the regulation of gene expression. However, the molecular mechanisms of hnRNP K reduction and its roles during apoptosis are still not understood. In the present study, we found that, following the appearance of the ΔPKC-δ, hnRNP K protein was significantly down-regulated in NSC606985, doxorubicin, arsenic trioxide and ultraviolet-induced apoptosis. We further provided evidence that ΔPKC-δ mediated the down-regulation of hnRNP K protein during apoptosis: PKC-δ inhibitor could rescue the reduction of hnRNP K; hnRNP K failed to be decreased in PKC-δ-deficient apoptotic KG1a cells; conditional induction of ΔPKC-δ in U937T cells directly down-regulated hnRNP K protein. Moreover, the proteasome inhibitor also inhibited the down-regulation of hnRNP K protein by apoptosis inducer and the conditional expression of ΔPKC-δ. More intriguingly, the suppression of hnRNP K with siRNA transfection significantly induced apoptosis. To our knowledge, this is the first demonstration that proteolytically activated PKC-δ down-regulates hnRNP K protein in a proteasome-dependent manner, which plays an important role in apoptosis induction.

  4. AMP-activated protein kinase: a key regulator of energy balance with many roles in human disease.

    Science.gov (United States)

    Grahame Hardie, D

    2014-12-01

    The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that regulates cellular and whole-body energy balance. A recently reported crystal structure has illuminated the complex regulatory mechanisms by which AMP and ADP cause activation of AMPK, involving phosphorylation by the upstream kinase LKB1. Once activated by falling cellular energy status, AMPK activates catabolic pathways that generate ATP whilst inhibiting anabolic pathways and other cellular processes that consume ATP. A role of AMPK is implicated in many human diseases. Mutations in the γ2 subunit cause heart disease due to excessive glycogen storage in cardiac myocytes, leading to ventricular pre-excitation. AMPK-activating drugs reverse many of the metabolic defects associated with insulin resistance, and recent findings suggest that the insulin-sensitizing effects of the widely used antidiabetic drug metformin are mediated by AMPK. The upstream kinase LKB1 is a tumour suppressor, and AMPK may exert many of its antitumour effects. AMPK activation promotes the oxidative metabolism typical of quiescent cells, rather than the aerobic glycolysis observed in tumour cells and cells involved in inflammation, explaining in part why AMPK activators have both antitumour and anti-inflammatory effects. Salicylate (the major in vivo metabolite of aspirin) activates AMPK, and this could be responsible for at least some of the anticancer and anti-inflammatory effects of aspirin. In addition to metformin and salicylates, novel drugs that modulate AMPK are likely to enter clinical trials soon. Finally, AMPK may be involved in viral infection: downregulation of AMPK during hepatitis C virus infection appears to be essential for efficient viral replication. PMID:24824502

  5. Cell division in the unicellular microalga Dunaliella viridis depends on phosphorylation of extracellular signal-regulated kinases (ERKs).

    Science.gov (United States)

    Jiménez, Carlos; Cossío, Belén R; Rivard, Christopher J; Berl, Tomás; Capasso, Juan M

    2007-01-01

    In mammalian cells, MAPKs are involved in both stress response (JNK and p38 pathways) and cell proliferation and differentiation [extracellular signal-regulated kinase (ERK)] through protein kinase cascades. Exposure of Dunaliella viridis cell cultures to PD98059, a very specific inhibitor of the ERK signalling pathway, resulted in a total arrest of cell proliferation and a complete dephosphorylation of ERK. As shown by flow cytometry analysis of propidium iodide-stained cells, PD98059 stopped mitosis at the G(2) phase after the S phase has been completed. Multiple physiological parameters such as cell motility and reducing power generation (NADPH) clearly indicate that the treated cells are wholly viable. Exposure of D. viridis to environmental stresses that impair cell division, such as hyperosmotic shock, nitrogen starvation, or sublethal UV irradiation, caused a marked decrease in the phospho-ERK levels as detected by western blot. Two 400 bp polynucleotides from D. viridis with high homologies to published sequences of ERK1 and ERK2 were cloned, sequenced, and submitted to GenBank. Northern blot analysis revealed two mRNA bands of approximately 1.9 kb, consistent with the expected size of ERK proteins ( approximately 40 kDa). Sequence analysis showed that they contained several mitogen-activated protein kinase (MAPK) conserved domains, including II, III, VIb, VII, and the double phosphorylation motif. Interestingly, in D. viridis, this motif was T*DY* instead of the canonic T*EY*. Based on this finding, ERK plant sequences can be divided into two groups, one termed the T*DY* branch and the other termed the T*EY* branch. The molecular and functional data presented here suggest that ERK is a very ancient signalling pathway and that it was already present in the last common ancestor of all eukaryotic cells. PMID:17220513

  6. Activation of ULK Kinase and Autophagy by GABARAP Trafficking from the Centrosome Is Regulated by WAC and GM130.

    Science.gov (United States)

    Joachim, Justin; Jefferies, Harold B J; Razi, Minoo; Frith, David; Snijders, Ambrosius P; Chakravarty, Probir; Judith, Delphine; Tooze, Sharon A

    2015-12-17

    Starvation-induced autophagy requires activation of the ULK complex at the phagophore. Two Golgi proteins, WAC and GM130, regulate autophagy, however their mechanism of regulation is unknown. In search of novel interaction partners of WAC, we found that GM130 directly interacts with WAC, and this interaction is required for autophagy. WAC is bound to the Golgi by GM130. WAC and GM130 interact with the Atg8 homolog GABARAP and regulate its subcellular localization. GABARAP is on the pericentriolar matrix, and this dynamic pool contributes to autophagosome formation. Tethering of GABARAP to the Golgi by GM130 inhibits autophagy, demonstrating an unexpected role for a golgin. WAC suppresses GM130 binding to GABARAP, regulating starvation-induced centrosomal GABARAP delivery to the phagophore. GABARAP, unlipidated and lipidated, but not LC3B, GABARAPL1, and GATE-16, specifically promotes ULK kinase activation dependent on the ULK1 LIR motif, elucidating a unique non-hierarchical role for GABARAP in starvation-induced activation of autophagy. PMID:26687599

  7. Cyclin-dependent kinase inhibitor p21 controls adult neural stem cell expansion by regulating Sox2 gene expression.

    Science.gov (United States)

    Marqués-Torrejón, M Ángeles; Porlan, Eva; Banito, Ana; Gómez-Ibarlucea, Esther; Lopez-Contreras, Andrés J; Fernández-Capetillo, Oscar; Vidal, Anxo; Gil, Jesús; Torres, Josema; Fariñas, Isabel

    2013-01-01

    In the adult brain, continual neurogenesis of olfactory neurons is sustained by the existence of neural stem cells (NSCs) in the subependymal niche. Elimination of the cyclin-dependent kinase inhibitor 1A (p21) leads to premature exhaustion of the subependymal NSC pool, suggesting a relationship between cell cycle control and long-term self-renewal, but the molecular mechanisms underlying NSC maintenance by p21 remain unexplored. Here we identify a function of p21 in the direct regulation of the expression of pluripotency factor Sox2, a key regulator of the specification and maintenance of neural progenitors. We observe that p21 directly binds a Sox2 enhancer and negatively regulates Sox2 expression in NSCs. Augmented levels of Sox2 in p21 null cells induce replicative stress and a DNA damage response that leads to cell growth arrest mediated by increased levels of p19(Arf) and p53. Our results show a regulation of NSC expansion driven by a p21/Sox2/p53 axis.

  8. Protein kinase C betaII regulates Akt phosphorylation on Ser-473 in a cell type- and stimulus-specific fashion.

    Science.gov (United States)

    Kawakami, Yuko; Nishimoto, Hajime; Kitaura, Jiro; Maeda-Yamamoto, Mari; Kato, Roberta M; Littman, Dan R; Leitges, Michael; Rawlings, David J; Kawakami, Toshiaki

    2004-11-12

    Akt (= protein kinase B), a subfamily of the AGC serine/threonine kinases, plays critical roles in survival, proliferation, glucose metabolism, and other cellular functions. Akt activation requires the recruitment of the enzyme to the plasma membrane by interacting with membrane-bound lipid products of phosphatidylinositol 3-kinase. Membrane-bound Akt is then phosphorylated at two sites for its full activation; Thr-308 in the activation loop of the kinase domain is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 in the C-terminal hydrophobic motif by a putative kinase PDK2. The identity of PDK2 has been elusive. Here we present evidence that conventional isoforms of protein kinase C (PKC), particularly PKCbetaII, can regulate Akt activity by directly phosphorylating Ser-473 in vitro and in IgE/antigen-stimulated mast cells. By contrast, PKCbeta is not required for Ser-473 phosphorylation in mast cells stimulated with stem cell factor or interleukin-3, in serum-stimulated fibroblasts, or in antigen receptor-stimulated T or B lymphocytes. Therefore, PKCbetaII appears to work as a cell type- and stimulus-specific PDK2. PMID:15364915

  9. G-patch domain and KOW motifs-containing protein, GPKOW; a nuclear RNA-binding protein regulated by protein kinase A

    OpenAIRE

    2011-01-01

    Background: Post-transcriptional processing of pre-mRNA takes place in several steps and requires involvement of a number of RNA-binding proteins. How pre-mRNA processing is regulated is in large enigmatic. The catalytic (C) subunit of protein kinase A (PKA) is a serine/threonine kinase, which regulates numerous cellular processes including pre-mRNA splicing. Despite that a significant fraction of the C subunit is found in splicing factor compartments in the nucleus, there are no indications ...

  10. The tomato NBARC-LRR protein Prf interacts with Pto kinase in vivo to regulate specific plant immunity.

    Science.gov (United States)

    Mucyn, Tatiana S; Clemente, Alfonso; Andriotis, Vasilios M E; Balmuth, Alexi L; Oldroyd, Giles E D; Staskawicz, Brian J; Rathjen, John P

    2006-10-01

    Immunity in tomato (Solanum lycopersicum) to Pseudomonas syringae bacteria expressing the effector proteins AvrPto and AvrPtoB requires both Pto kinase and the NBARC-LRR (for nucleotide binding domain shared by Apaf-1, certain R gene products, and CED-4 fused to C-terminal leucine-rich repeats) protein Prf. Pto plays a direct role in effector recognition within the host cytoplasm, but the role of Prf is unknown. We show that Pto and Prf are coincident in the signal transduction pathway that controls ligand-independent signaling. Pto and Prf associate in a coregulatory interaction that requires Pto kinase activity and N-myristoylation for signaling. Pto interacts with a unique Prf N-terminal domain outside of the NBARC-LRR domain and resides in a high molecular weight recognition complex dependent on the presence of Prf. In this complex, both Pto and Prf contribute to specific recognition of AvrPtoB. The data suggest that the role of Pto is confined to the regulation of Prf and that the bacterial effectors have evolved to target this coregulatory molecular switch.

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

    Directory of Open Access Journals (Sweden)

    Shen-Hsi Yang

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

  12. A Cyclic di-GMP-binding Adaptor Protein Interacts with Histidine Kinase to Regulate Two-component Signaling.

    Science.gov (United States)

    Xu, Linghui; Venkataramani, Prabhadevi; Ding, Yichen; Liu, Yang; Deng, Yinyue; Yong, Grace Lisi; Xin, Lingyi; Ye, Ruijuan; Zhang, Lianhui; Yang, Liang; Liang, Zhao-Xun

    2016-07-29

    The bacterial messenger cyclic di-GMP (c-di-GMP) binds to a diverse range of effectors to exert its biological effect. Despite the fact that free-standing PilZ proteins are by far the most prevalent c-di-GMP effectors known to date, their physiological function and mechanism of action remain largely unknown. Here we report that the free-standing PilZ protein PA2799 from the opportunistic pathogen Pseudomonas aeruginosa interacts directly with the hybrid histidine kinase SagS. We show that PA2799 (named as HapZ: histidine kinase associated PilZ) binds directly to the phosphoreceiver (REC) domain of SagS, and that the SagS-HapZ interaction is further enhanced at elevated c-di-GMP concentration. We demonstrate that binding of HapZ to SagS inhibits the phosphotransfer between SagS and the downstream protein HptB in a c-di-GMP-dependent manner. In accordance with the role of SagS as a motile-sessile switch and biofilm growth factor, we show that HapZ impacts surface attachment and biofilm formation most likely by regulating the expression of a large number of genes. The observations suggest a previously unknown mechanism whereby c-di-GMP mediates two-component signaling through a PilZ adaptor protein.

  13. Class I PI-3-Kinase Signaling Is Critical for Bone Formation Through Regulation of SMAD1 Activity in Osteoblasts.

    Science.gov (United States)

    Gámez, Beatriz; Rodríguez-Carballo, Edgardo; Graupera, Mariona; Rosa, José Luis; Ventura, Francesc

    2016-08-01

    Bone formation and homeostasis is carried out by osteoblasts, whose differentiation and activity are regulated by osteogenic signaling networks. A central mediator of these inputs is the lipid kinase phosphatidylinositol 3-kinase (PI3K). However, at present, there are no data on the specific role of distinct class IA PI3K isoforms in bone biology. Here, we performed osteoblast-specific deletion in mice to show that both p110α and p110β isoforms are required for survival and differentiation and function of osteoblasts and thereby control bone formation and postnatal homeostasis. Impaired osteogenesis arises from increased GSK3 activity and a depletion of SMAD1 protein levels in PI3K-deficient osteoblasts. Accordingly, pharmacological inhibition of GSK3 activity or ectopic expression of SMAD1 or SMAD5 normalizes bone morphogenetic protein (BMP) transduction and osteoblast differentiation. Together, these results identify the PI3K-GSK3-SMAD1 axis as a central node integrating multiple signaling networks that govern bone formation and homeostasis. © 2016 American Society for Bone and Mineral Research. PMID:26896753

  14. Cyclosporin A does not block the phorbol ester - protein kinase C regulated pathway of T cell activation

    International Nuclear Information System (INIS)

    The T cell line Jurkat can be induced to produce interleukin-2 (IL-2) in vitro by a combination of two stimuli: (1) A stimulus that increases cytoplasmic free Ca++ concentration plus (2) phorbol ester (PMA). No. IL-2 production is induced with either stimulus alone. The T cell line HUT 78 responds to the same combination of stimuli, however also produces low amounts of IL-2 in response to PMA only. After HUT 78 cells were pretreated with the nucleoside analog 5-azacytidine (AZA) they produced maximal amounts of IL-2 in response to PMA alone. Cyclosporin A (CsA) has been shown to completely block the two stimulus-induced IL-2 production in Jurkat at a pretranslational level. In contrast, the low level of IL-2 production in HUT 78 and the high level of IL-2 production in AZA-treated HUT 78 induced by PMA only is not inhibited by CsA. Additionally we demonstrated that CsA did not inhibit activation of protein kinase C, the primary target enzyme in PMA induced cell activation. The presented data suggest that CsA does not globally block lymphokine expression but rather interferes with signaling events in T cell activation. It appears that CsA blocks the pathway controlled by either Ca++ alone or Ca++ in combination with PMA, but not activation signaling regulated by PMA induced activation of protein kinase C alone

  15. A novel hybrid kinase is essential for regulating the sigma(B)-mediated stress response of Bacillus cereus.

    Science.gov (United States)

    de Been, Mark; Tempelaars, Marcel H; van Schaik, Willem; Moezelaar, Roy; Siezen, Roland J; Abee, Tjakko

    2010-03-01

    A common bacterial strategy for monitoring environmental challenges is to use two-component systems, which consist of a sensor histidine kinase (HK) and a response regulator (RR). In the food-borne pathogen Bacillus cereus, the alternative sigma factor sigma(B) is activated by the RR RsbY. Here we present strong indications that the PP2C-type phosphatase RsbY receives its input from the multi-sensor hybrid kinase BC1008 (renamed RsbK). Genome analyses revealed that, across bacilli, rsbY and rsbK are located in a conserved gene cluster. A B. cereus rsbK deletion strain was shown to be incapable of inducing sigma(B) upon stress conditions and was impaired in its heat adaptive response. Comparison of the wild-type and rsbK mutant transcriptomes upon heat shock revealed that RsbK was primarily involved in the activation of the sigma(B)-mediated stress response. Truncation of the RsbK RR receiver domain demonstrated the importance of this domain for sigma(B) induction upon stress. The domain architecture of RsbK suggests that in the B. cereus group and in other bacilli, environmental and intracellular stress signalling routes are combined into one single protein. This strategy is markedly different from the sigma(B) activation pathway in other low-GC Gram-positives.

  16. A Cyclic di-GMP-binding Adaptor Protein Interacts with Histidine Kinase to Regulate Two-component Signaling.

    Science.gov (United States)

    Xu, Linghui; Venkataramani, Prabhadevi; Ding, Yichen; Liu, Yang; Deng, Yinyue; Yong, Grace Lisi; Xin, Lingyi; Ye, Ruijuan; Zhang, Lianhui; Yang, Liang; Liang, Zhao-Xun

    2016-07-29

    The bacterial messenger cyclic di-GMP (c-di-GMP) binds to a diverse range of effectors to exert its biological effect. Despite the fact that free-standing PilZ proteins are by far the most prevalent c-di-GMP effectors known to date, their physiological function and mechanism of action remain largely unknown. Here we report that the free-standing PilZ protein PA2799 from the opportunistic pathogen Pseudomonas aeruginosa interacts directly with the hybrid histidine kinase SagS. We show that PA2799 (named as HapZ: histidine kinase associated PilZ) binds directly to the phosphoreceiver (REC) domain of SagS, and that the SagS-HapZ interaction is further enhanced at elevated c-di-GMP concentration. We demonstrate that binding of HapZ to SagS inhibits the phosphotransfer between SagS and the downstream protein HptB in a c-di-GMP-dependent manner. In accordance with the role of SagS as a motile-sessile switch and biofilm growth factor, we show that HapZ impacts surface attachment and biofilm formation most likely by regulating the expression of a large number of genes. The observations suggest a previously unknown mechanism whereby c-di-GMP mediates two-component signaling through a PilZ adaptor protein. PMID:27231351

  17. Protein Kinase D1-Dependent Phosphorylation of Dopamine D1 Receptor Regulates Cocaine-Induced Behavioral Responses

    Science.gov (United States)

    Wang, Ning; Su, Ping; Zhang, Ying; Lu, Jie; Xing, Baoming; Kang, Kai; Li, Wenqi; Wang, Yun

    2014-01-01

    The dopamine (DA) D1 receptor (D1R) is critically involved in reward and drug addiction. Phosphorylation-mediated desensitization or internalization of D1R has been extensively investigated. However, the potential for upregulation of D1R function through phosphorylation remains to be determined. Here we report that acute cocaine exposure induces protein kinase D1 (PKD1) activation in the rat striatum, and knockdown of PKD1 in the rat dorsal striatum attenuates cocaine-induced locomotor hyperactivity. Moreover, PKD1-mediated phosphorylation of serine 421 (S421) of D1R promotes surface localization of D1R and enhances downstream extracellular signal-regulated kinase signaling in D1R-transfected HEK 293 cells. Importantly, injection of the peptide Tat-S421, an engineered Tat fusion-peptide targeting S421 (Tat-S421), into the rat dorsal striatum inhibits cocaine-induced locomotor hyperactivity and injection of Tat-S421 into the rat hippocampus or the shell of the nucleus accumbens (NAc) also inhibits cocaine-induced conditioned place preference (CPP). However, injection of Tat-S421 into the rat NAc shell does not establish CPP by itself and injection of Tat-S421 into the hippocampus does not influence spatial learning and memory. Thus, targeting S421 of D1R represents a promising strategy for the development of pharmacotherapeutic treatments for drug addiction and other disorders that result from DA imbalances. PMID:24362306

  18. Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle

    DEFF Research Database (Denmark)

    Treebak, Jonas Thue; Taylor, Eric B.; Witczak, Carol A.;

    2010-01-01

    . The majority of known phosphorylation sites on TBC1D4 lie within the Akt consensus motif and are phosphorylated by insulin stimulation. However, the 5 AMP activated protein kinase (AMPK) and other kinases may also phosphorylate TBC1D4, and therefore we hypothesized the presence of additional phosphorylation......TBC1D4 (also known as AS160) regulates GLUT4 translocation and glucose uptake in adipocytes and skeletal muscle. Its mode of action involves phosphorylation of Serine (S)/Threonine (T) residues by upstream kinases resulting in inactivation of Rab-GAP activity leading to GLUT4 mobilization...... that S711 would be recognized by AMPK. Using a phospho-specific antibody against S711, we found that AICAR and contraction increased S711 phosphorylation in mouse skeletal muscle and this increase was abolished in muscle-specific AMPKalpha2 kinase dead transgenic mice. Exercise in human vastus lateralis...

  19. Both mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) 1/2 and phosphatidylinositide-3-OH kinase (PI3K)/Akt pathways regulate activation of E-twenty-six (ETS)-like transcription factor 1 (Elk-1) in U138 glioblastoma cells.

    Science.gov (United States)

    Mut, Melike; Lule, Sevda; Demir, Ozlem; Kurnaz, Isil Aksan; Vural, Imran

    2012-02-01

    Epidermal growth factor (EGF) and its receptor (EGFR) have been shown to play a significant role in the pathogenesis of glioblastoma. In our study, the EGFR was stimulated with EGF in human U138 glioblastoma cells. We show that the activated mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) 1/2 pathway phosphorylated the E twenty-six (ETS)-like transcription factor 1 (Elk-1) mainly at serine 383 residue. Mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, UO126 and ERK inhibitor II, FR180204 blocked the Elk-1 phosphorylation and activation. The phosphatidylinositide-3-OH kinase (PI3K)/Akt pathway was also involved in the Elk-1 activation. Activation of the Elk-1 led to an increased survival and a proliferative response with the EGF stimulation in the U138 glioblastoma cells. Knocking-down the Elk-1 using an RNA interference technique caused a decrease in survival of the unstimulated U138 glioblastoma cells and also decreased the proliferative response to the EGF stimulation. The Elk-1 transcription factor was important for the survival and proliferation of U138 glioblastoma cells upon the stimulation of EGFR with EGF. The MAPK/ERK1/2 and PI3K/Akt pathways regulated this response via activation of the Elk-1 transcription factor. The Elk-1 may be one of the convergence points for pathways located downstream of EGFR in glioblastoma cells. Utilization of the Elk-1 as a therapeutic target may lead to a novel strategy in treatment of glioblastoma.

  20. Up-regulated uridine kinase gene identified by RLCS in the ventral horn after crush injury to rat sciatic nerves.

    Science.gov (United States)

    Yuh, I; Yaoi, T; Watanabe, S; Okajima, S; Hirasawa, Y; Fushiki, S

    1999-12-01

    Rat sciatic nerve crush injury is one of the models commonly employed for studying the mechanisms of nerve regeneration. In this study, we analyzed the temporal change of gene expression after injury in this model, to elucidate the molecular mechanisms involved in nerve regeneration. First, a cDNA analysis method, Restriction Landmark cDNA Scanning (RLCS), was applied to cells in the ventral horn of the spinal cord during a 7-day period after the crush injury. A total of 1991 cDNA species were detected as spots on gels, and 37 of these were shown to change after the injury. Temporally changed patterns were classified into three categories: the continuously up-regulated type (10 species), the transiently up-regulated type (22 species), and the down-regulated type (5 species). These complex patterns of gene expression demonstrated after the injury suggest that precise regulation in molecular pathways is required for accomplishing nerve regeneration. Secondly, the rat homologue of uridine kinase gene was identified as one of the up-regulated genes. Northern blot analysis on rat ventral horn tissue and brain revealed that the UK gene had three transcripts with different sizes (4.3, 1. 4, and 1.35 kb, respectively). All of the transcripts, especially the 4.3 kb one, were up-regulated mainly in a bimodal fashion during the 28-day period after the injury. The RLCS method that we employed in the present study shows promise as a means to fully analyze molecular changes in nerve regeneration in detail. PMID:10581173

  1. The kinase regulator mob1 acts as a patterning protein for stentor morphogenesis.

    Directory of Open Access Journals (Sweden)

    Mark M Slabodnick

    2014-05-01

    Full Text Available Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain largely unknown. Although all cells develop patterns, they are most obvious in ciliates; hence, we have turned to a classical unicellular model system, the giant ciliate Stentor coeruleus. Here we show that the RNA interference (RNAi machinery is conserved in Stentor. Using RNAi, we identify the kinase coactivator Mob1--with conserved functions in cell division and morphogenesis from plants to humans-as an asymmetrically localized patterning protein required for global patterning during development and regeneration in Stentor. Our studies reopen the door for Stentor as a model regeneration system.

  2. Design and synthesis of orally bioavailable serum and glucocorticoid-regulated kinase 1 (SGK1) inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Hammond, Marlys; Washburn, David G.; Hoang, Tram H.; Manns, Sharada; Frazee, James S.; Nakamura, Hiroko; Patterson, Jaclyn R.; Trizna, Walter; Wu, Charlene; Azzarano, Leonard M.; Nagilla, Rakesh; Nord, Melanie; Trejo, Rebecca; Head, Martha S.; Zhao, Baoguang; Smallwood, Angela M.; Hightower, Kendra; Laping, Nicholas J.; Schnackenberg, Christine G.; Thompson, Scott K.; (GSKPA)

    2010-09-27

    The lead serum and glucocorticoid-related kinase 1 (SGK1) inhibitors 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (1) and {l_brace}4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl{r_brace}acetic acid (2) suffer from low DNAUC values in rat, due in part to formation and excretion of glucuronic acid conjugates. These PK/glucuronidation issues were addressed either by incorporating a substituent on the 3-phenyl ring ortho to the key carboxylate functionality of 1 or by substituting on the group in between the carboxylate and phenyl ring of 2. Three of these analogs have been identified as having good SGK1 inhibition potency and have DNAUC values suitable for in vivo testing.

  3. Design and synthesis of orally bioavailable serum and glucocorticoid-regulated kinase 1 (SGK1) inhibitors.

    Science.gov (United States)

    Hammond, Marlys; Washburn, David G; Hoang, H Tram; Manns, Sharada; Frazee, James S; Nakamura, Hiroko; Patterson, Jaclyn R; Trizna, Walter; Wu, Charlene; Azzarano, Leonard M; Nagilla, Rakesh; Nord, Melanie; Trejo, Rebecca; Head, Martha S; Zhao, Baoguang; Smallwood, Angela M; Hightower, Kendra; Laping, Nicholas J; Schnackenberg, Christine G; Thompson, Scott K

    2009-08-01

    The lead serum and glucocorticoid-related kinase 1 (SGK1) inhibitors 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (1) and {4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid (2) suffer from low DNAUC values in rat, due in part to formation and excretion of glucuronic acid conjugates. These PK/glucuronidation issues were addressed either by incorporating a substituent on the 3-phenyl ring ortho to the key carboxylate functionality of 1 or by substituting on the group in between the carboxylate and phenyl ring of 2. Three of these analogs have been identified as having good SGK1 inhibition potency and have DNAUC values suitable for in vivo testing.

  4. Involvement of extracellular signal-regulated kinase/mitogen activated protein kinase pathway in multidrug resistance induced by HBx in hepatoma cell line

    Institute of Scientific and Technical Information of China (English)

    Jian Guan; Xiao-Ping Chen; Hong Zhu; Shun-Feng Luo; Bin Cao; Lei Ding

    2004-01-01

    AIM: To investigate the molecular mechanism of the influence of HBx protein on multidrug resistance associated genes:multidrug resistance 1 (MDR-1), multidrug related protein (MRP-1), lung resistance related protein (LRP) in hepatoma cells and the potential role of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in this process.METHODS: A cell model stably expressing the HBx protein was established by liposome-mediated transfection of HBx gene into HepG2 cell line. The expression of multidrug resistance associated genes and proteins was detected by RT-PCR and Western blot. AnnexinV-FITC/PI assay was used to confirm the multidrug resistance (MDR) phenotype of transfected cells by fluorescence cytometry (FACS). The ERK/MAPK pathway activation was measured by Western blot through comparing the ratio of phosphorylation of ERK/MAPK to total ERK/MAPK protein. After treated with the ERK/MAPK pathway inhibitor U0126, the HBx-expressing cells were harvested. Then RT-PCR, Western blot and FACS were used to analyze the alterations in the expression of multidrug resistance associated genes and the MDR phenotype after exposure.RESULTS: Compared with the control group, the transfected cells showed a higher expression of MDR associated genes and proteins. Marked elevations in MDR-1 (64.3%), MRP-1 (87.5%) and LRP (90.8%) were observed in the transfected cells (P<0.05). RT-PCR revealed that the over-expression of MDR associated proteins was due to amplification of such genes (MDR1 2.9 fold, MRP1 1.67 fold, LRP1.95 fold).Furthermore, we found that the ERK/MAPK activity was remarkably high in the HBx-expressing cells. The activation of ERK/MAPK, as measured by the ratio of phosphorylated ERK bands normalized to the total ERK bands, was increased by 2.3-fold in HBx-transfected cells compared with cells transfected with the empty vector. After treated with the ERK/MAPK pathway inhibitor, the level of MDR associated genes and proteins in the

  5. Mitogen-activated protein kinase (MAPK pathway regulates branching by remodeling epithelial cell adhesion.

    Directory of Open Access Journals (Sweden)

    Anneliis Ihermann-Hella

    2014-03-01

    Full Text Available Although the growth factor (GF signaling guiding renal branching is well characterized, the intracellular cascades mediating GF functions are poorly understood. We studied mitogen-activated protein kinase (MAPK pathway specifically in the branching epithelia of developing kidney by genetically abrogating the pathway activity in mice lacking simultaneously dual-specificity protein kinases Mek1 and Mek2. Our data show that MAPK pathway is heterogeneously activated in the subset of G1- and S-phase epithelial cells, and its tissue-specific deletion results in severe renal hypodysplasia. Consequently to the deletion of Mek1/2, the activation of ERK1/2 in the epithelium is lost and normal branching pattern in mutant kidneys is substituted with elongation-only phenotype, in which the epithelium is largely unable to form novel branches and complex three-dimensional patterns, but able to grow without primary defects in mitosis. Cellular characterization of double mutant epithelium showed increased E-cadherin at the cell surfaces with its particular accumulation at baso-lateral locations. This indicates changes in cellular adhesion, which were revealed by electron microscopic analysis demonstrating intercellular gaps and increased extracellular space in double mutant epithelium. When challenged to form monolayer cultures, the mutant epithelial cells were impaired in spreading and displayed strong focal adhesions in addition to spiky E-cadherin. Inhibition of MAPK activity reduced paxillin phosphorylation on serine 83 while remnants of phospho-paxillin, together with another focal adhesion (FA protein vinculin, were augmented at cell surface contacts. We show that MAPK activity is required for branching morphogenesis, and propose that it promotes cell cycle progression and higher cellular motility through remodeling of cellular adhesions.

  6. The protein kinase D1 COOH terminus: marker or regulator of enzyme activity?

    Science.gov (United States)

    Qiu, Weihua; Zhang, Fan; Steinberg, Susan F

    2014-10-01

    Protein kinase D1 (PKD1) is a Ser/Thr kinase implicated in a wide variety of cellular responses. PKD1 activation is generally attributed to a PKC-dependent pathway that leads to phosphorylation of the activation loop at Ser(744)/Ser(748). This modification increases catalytic activity, including that toward an autophosphorylation site (Ser(916)) in a postsynaptic density-95/disks large/zonula occludens-1 (PDZ)-binding motif at the extreme COOH terminus. However, there is growing evidence that PKD1 activation can also result from a PKC-independent autocatalytic reaction at Ser(744)/Ser(748) and that certain stimuli increase in PKD1 phosphorylation at Ser(744)/S(748) without an increase in autophosphorylation at Ser(916). This study exposes a mechanism that results in a discrepancy between PKD1 COOH-terminal autocatalytic activity and activity toward other substrates. We show that PKD1 constructs harboring COOH-terminal epitope tags display high levels of in vitro activation loop autocatalytic activity and activity toward syntide-2 (a peptide substrate), but no Ser(916) autocatalytic activity. Cell-based studies show that the COOH-terminal tag, adjacent to PKD1's PDZ1-binding motif, does not grossly influence PKD1 partitioning between soluble and particulate fractions in resting cells or PKD1 translocation to the particulate fraction following treatment with PMA. However, a COOH-terminal tag that confers a high level of activation loop autocatalytic activity decreases the PKC requirement for agonist-dependent PKD1 activation in cells. The recognition that COOH-terminal tags alter PKD1's pharmacological profile is important from a technical standpoint. The altered dynamics and activation mechanisms for COOH-terminal-tagged PKD1 enzymes also could model the signaling properties of localized pools of enzyme anchored through the COOH terminus to PDZ domain-containing scaffolding proteins.

  7. Intracellular Theileria annulata promote invasive cell motility through kinase regulation of the host actin cytoskeleton.

    Directory of Open Access Journals (Sweden)

    Min Ma

    2014-03-01

    Full Text Available The intracellular, protozoan Theileria species parasites are the only eukaryotes known to transform another eukaryotic cell. One consequence of this parasite-dependent transformation is the acquisition of motile and invasive properties of parasitized cells in vitro and their metastatic dissemination in the animal, which causes East Coast Fever (T. parva or Tropical Theileriosis (T. annulata. These motile and invasive properties of infected host cells are enabled by parasite-dependent, poorly understood F-actin dynamics that control host cell membrane protrusions. Herein, we dissected functional and structural alterations that cause acquired motility and invasiveness of T. annulata-infected cells, to understand the molecular basis driving cell dissemination in Tropical Theileriosis. We found that chronic induction of TNFα by the parasite contributes to motility and invasiveness of parasitized host cells. We show that TNFα does so by specifically targeting expression and function of the host proto-oncogenic ser/thr kinase MAP4K4. Blocking either TNFα secretion or MAP4K4 expression dampens the formation of polar, F-actin-rich invasion structures and impairs cell motility in 3D. We identified the F-actin binding ERM family proteins as MAP4K4 downstream effectors in this process because TNFα-induced ERM activation and cell invasiveness are sensitive to MAP4K4 depletion. MAP4K4 expression in infected cells is induced by TNFα-JNK signalling and maintained by the inhibition of translational repression, whereby both effects are parasite dependent. Thus, parasite-induced TNFα promotes invasive motility of infected cells through the activation of MAP4K4, an evolutionary conserved kinase that controls cytoskeleton dynamics and cell motility. Hence, MAP4K4 couples inflammatory signaling to morphodynamic processes and cell motility, a process exploited by the intracellular Theileria parasite to increase its host cell's dissemination capabilities.

  8. 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

  9. Adenylate kinase from Streptococcus pneumoniae is essential for growth through its catalytic activity

    Directory of Open Access Journals (Sweden)

    Trung Thanh Thach

    2014-01-01

    Full Text Available Streptococcus pneumoniae (pneumococcus infection causes more than 1.6 million deaths worldwide. Pneumococcal growth is a prerequisite for its virulence and requires an appropriate supply of cellular energy. Adenylate kinases constitute a major family of enzymes that regulate cellular ATP levels. Some bacterial adenylate kinases (AdKs are known to be critical for growth, but the physiological effects of AdKs in pneumococci have been poorly understood at the molecular level. Here, by crystallographic and functional studies, we report that the catalytic activity of adenylate kinase from S. pneumoniae (SpAdK serotype 2 D39 is essential for growth. We determined the crystal structure of SpAdK in two conformations: ligand-free open form and closed in complex with a two-substrate mimic inhibitor adenosine pentaphosphate (Ap5A. Crystallographic analysis of SpAdK reveals Arg-89 as a key active site residue. We generated a conditional expression mutant of pneumococcus in which the expression of the adk gene is tightly regulated by fucose. The expression level of adk correlates with growth rate. Expression of the wild-type adk gene in fucose-inducible strains rescued a growth defect, but expression of the Arg-89 mutation did not. SpAdK increased total cellular ATP levels. Furthermore, lack of functional SpAdK caused a growth defect in vivo. Taken together, our results demonstrate that SpAdK is essential for pneumococcal growth in vitro and in vivo.

  10. The Gln223Arg polymorphism of the leptin receptor in Pima Indians: influence on energy expenditure, physical activity and lipid metabolism

    DEFF Research Database (Denmark)

    Stefan, N; Vozarova, B; Del Parigi, A;

    2002-01-01

    Leptin regulates body weight by its receptor-mediated anorectic, thermogenic and antisteatotic effects. Recently, lower leptin binding to the soluble form of the leptin receptor (LEPR) was shown in carriers of the Arg223-encoding allele of the Gln223Arg polymorphism of the LEPR. To investigate...

  11. The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth.

    Science.gov (United States)

    Turapov, Obolbek; Loraine, Jessica; Jenkins, Christopher H; Barthe, Philippe; McFeely, Daniel; Forti, Francesca; Ghisotti, Daniela; Hesek, Dusan; Lee, Mijoon; Bottrill, Andrew R; Vollmer, Waldemar; Mobashery, Shahriar; Cohen-Gonsaud, Martin; Mukamolova, Galina V

    2015-07-01

    PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to β-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth of Mycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture.

  12. FancJ regulates interstrand crosslinker induced centrosome amplification through the activation of polo-like kinase 1

    Directory of Open Access Journals (Sweden)

    Jianqiu Zou

    2013-08-01

    DNA damage response (DDR and the centrosome cycle are two of the most critical processes for maintaining a stable genome in animals. Sporadic evidence suggests a connection between these two processes. Here, we report our findings that six Fanconi Anemia (FA proteins, including FancI and FancJ, localize to the centrosome. Intriguingly, we found that the localization of FancJ to the mother centrosome is stimulated by a DNA interstrand crosslinker, Mitomycin C (MMC. We further show that, in addition to its role in interstrand crosslinking (ICL repair, FancJ also regulates the normal centrosome cycle as well as ICL induced centrosome amplification by activating the polo-like kinase 1 (PLK1. We have uncovered a novel function of FancJ in centrosome biogenesis and established centrosome amplification as an integral part of the ICL response.

  13. Arg-Phe-amide-related peptides influence gonadotropin-releasing hormone neurons

    Institute of Scientific and Technical Information of China (English)

    Haluk Kelestimur; Emine Kacar; Aysegul Uzun; Mete Ozcan; Selim Kutlu

    2013-01-01

    The hypothalamic Arg-Phe-amide-related peptides, gonadotropin-inhibitory hormone and orthologous mammalian peptides of Arg-Phe-amide, may be important regulators of the hypothalamus-pituitary-gonadal reproductive axis. These peptides may modulate the effects of kisspeptins because they are presently recognized as the most potent activators of the hypothalamus-pituitary-gonadal axis. However, their effects on gonadotropin-releasing hormone neurons have not been investigated. In the current study, the GT1–7 cell line-expressing gonadotropin-releasing hormone was used as a model to explore the effects of Arg-Phe- amide-related peptides on kisspeptin activation. Intracellular calcium concentration was quantified using the calcium-sensitive dye, fura-2 acetoxymethyl ester. Gonadotropin-releasing hormone released into the medium was detected via enzyme-linked immunosorbent assay. Results showed that 100 nmol/L kisspeptin-10 significantly increased gonadotropin-releasing hormone levels (at 120 minutes of exposure) and intracellular calcium concentrations. Co-treatment of kisspeptin with 1 μmol/L gonadotropin-inhibitory hormone or 1 μmol/L Arg-Phe-amide-related peptide-1 significantly attenuated levels of kisspeptin-induced gonadotropin-releasing hormone but did not affect kisspeptin-induced elevations of intracellular calcium concentration. Overall, the results suggest that gonadotropin-inhibitory hormone and Arg-Phe-amide-related peptide-1 may have inhibitory effects on kisspeptin-activated gonadotropin-releasing hormone neurons independent of the calcium signaling pathway.

  14. Nuclear localization of lymphocyte-specific protein tyrosine kinase (Lck) and its role in regulating LIM domain only 2 (Lmo2) gene

    Energy Technology Data Exchange (ETDEWEB)

    Venkitachalam, Srividya; Chueh, Fu-Yu [Department of Microbiology and Immunology, H. M. Bligh Cancer Research Laboratories, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064 (United States); Yu, Chao-Lan, E-mail: chaolan.yu@rosalindfranklin.edu [Department of Microbiology and Immunology, H. M. Bligh Cancer Research Laboratories, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064 (United States)

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer Lmo2 expression is elevated in Lck-transformed cells. Black-Right-Pointing-Pointer Both endogenous and exogenous Lck localize in the nucleus. Black-Right-Pointing-Pointer Nuclear Lck is active in Lck-transformed cells. Black-Right-Pointing-Pointer Lck binds to the promoter region of Lmo2 gene in vivo. Black-Right-Pointing-Pointer In contrast to JAK2, Lck does not increase histone H3 phosphorylation on Tyr 41. -- Abstract: LIM domain only protein 2 (Lmo2) is a transcription factor that plays a critical role in the development of T-acute lymphoblastic leukemia (T-ALL). A previous report established a link between Lmo2 expression and the nuclear presence of oncogenic Janus kinase 2 (JAK2), a non-receptor protein tyrosine kinase. The oncogenic JAK2 kinase phosphorylates histone H3 on Tyr 41 that leads to the relief of Lmo2 promoter repression and subsequent gene expression. Similar to JAK2, constitutive activation of lymphocyte-specific protein tyrosine kinase (Lck) has been implicated in lymphoid malignancies. However, it is not known whether oncogenic Lck regulates Lmo2 expression through a similar mechanism. We show here that Lmo2 expression is significantly elevated in T cell leukemia LSTRA overexpressing active Lck kinase and in HEK 293 cells expressing oncogenic Y505FLck kinase. Nuclear localization of active Lck kinase was confirmed in both Lck-transformed cells by subcellular fractionation and immunofluorescence microscopy. More importantly, in contrast to oncogenic JAK2, oncogenic Lck kinase does not result in significant increase in histone H3 phosphorylation on Tyr 41. Instead, chromatin immunoprecipitation experiment shows that oncogenic Y505FLck kinase binds to the Lmo2 promoter in vivo. This result raises the possibility that oncogenic Lck may activate Lmo2 promoter through direct interaction.

  15. Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor α Kinase Activity.

    Science.gov (United States)

    Green, Jenna; Endale, Mehari; Auer, Herbert; Perl, Anne-Karina T

    2016-04-01

    Epithelial-mesenchymal cell interactions and factors that control normal lung development are key players in lung injury, repair, and fibrosis. A number of studies have investigated the roles and sources of epithelial progenitors during lung regeneration; such information, however, is limited in lung fibroblasts. Thus, understanding the origin, phenotype, and roles of fibroblast progenitors in lung development, repair, and regeneration helps address these limitations. Using a combination of platelet-derived growth factor receptor α-green fluorescent protein (PDGFRα-GFP) reporter mice, microarray, real-time polymerase chain reaction, flow cytometry, and immunofluorescence, we characterized two distinct interstitial resident fibroblasts, myo- and matrix fibroblasts, and identified a role for PDGFRα kinase activity in regulating their activation during lung regeneration. Transcriptional profiling of the two populations revealed a myo- and matrix fibroblast gene signature. Differences in proliferation, smooth muscle actin induction, and lipid content in the two subpopulations of PDGFRα-expressing fibroblasts during alveolar regeneration were observed. Although CD140α(+)CD29(+) cells behaved as myofibroblasts, CD140α(+)CD34(+) appeared as matrix and/or lipofibroblasts. Gain or loss of PDGFRα kinase activity using the inhibitor nilotinib and a dominant-active PDGFRα-D842V mutation revealed that PDGFRα was important for matrix fibroblast differentiation. We demonstrated that PDGFRα signaling promotes alveolar septation by regulating fibroblast activation and matrix fibroblast differentiation, whereas myofibroblast differentiation was largely PDGFRα independent. These studies provide evidence for the phenotypic and functional diversity as well as the extent of specificity of interstitial resident fibroblasts differentiation during regeneration after partial pneumonectomy. PMID:26414960

  16. Dbf4 regulates the Cdc5 Polo-like kinase through a distinct non-canonical binding interaction.

    Science.gov (United States)

    Chen, Ying-Chou; Weinreich, Michael

    2010-12-31

    Cdc7-Dbf4 is a conserved, two-subunit kinase required for initiating eukaryotic DNA replication. Recent studies have shown that Cdc7-Dbf4 also regulates the mitotic exit network (MEN) and monopolar homolog orientation in meiosis I (Matos, J., Lipp, J. J., Bogdanova, A., Guillot, S., Okaz, E., Junqueira, M., Shevchenko, A., and Zachariae, W. (2008) Cell 135, 662-678 and Miller, C. T., Gabrielse, C., Chen, Y. C., and Weinreich, M. (2009) PLoS Genet. 5, e1000498). Both activities likely involve a Cdc7-Dbf4 interaction with Cdc5, the single Polo-like kinase in budding yeast. We previously showed that Dbf4 binds the Cdc5 polo-box domain (PBD) via an ∼40-residue N-terminal sequence, which lacks a PBD consensus binding site (S(pS/pT)(P/X)), and that Dbf4 inhibits Cdc5 function during mitosis. Here we identify a non-consensus PBD binding site within Dbf4 and demonstrate that the PBD-Dbf4 interaction occurs via a distinct PBD surface from that used to bind phosphoproteins. Genetic and biochemical analysis of multiple dbf4 mutants indicate that Dbf4 inhibits Cdc5 function through direct binding. Surprisingly, mutation of invariant Cdc5 residues required for binding phosphorylated substrates has little effect on yeast viability or growth rate. Instead, cdc5 mutants defective for binding phosphoproteins exhibit enhanced resistance to microtubule disruption and an increased rate of spindle elongation. This study, therefore, details the molecular nature of a new type of PBD binding and reveals that Cdc5 targeting to phosphorylated substrates likely regulates spindle dynamics.

  17. Phosphoinositide 3-Kinase δ Regulates Dectin-2 Signaling and the Generation of Th2 and Th17 Immunity.

    Science.gov (United States)

    Lee, Min Jung; Yoshimoto, Eri; Saijo, Shinobu; Iwakura, Yoichiro; Lin, Xin; Katz, Howard R; Kanaoka, Yoshihide; Barrett, Nora A

    2016-07-01

    The C-type lectin receptor Dectin-2 can trigger the leukotriene C4 synthase-dependent generation of cysteinyl leukotrienes and the caspase-associated recruitment domain 9- and NF-κB-dependent generation of cytokines, such as IL-23, IL-6, and TNF-α, to promote Th2 and Th17 immunity, respectively. Dectin-2 activation also elicits the type 2 cytokine IL-33, but the mechanism by which Dectin-2 induces these diverse innate mediators is poorly understood. In this study, we identify a common upstream requirement for PI3Kδ activity for the generation of each Dectin-2-dependent mediator elicited by the house dust mite species, Dermatophagoides farinae, using both pharmacologic inhibition and small interfering RNA knockdown of PI3Kδ in bone marrow-derived dendritic cells. PI3Kδ activity depends on spleen tyrosine kinase (Syk) and regulates the activity of protein kinase Cδ, indicating that PI3Kδ is a proximal Syk-dependent signaling intermediate. Inhibition of PI3Kδ also reduces cysteinyl leukotrienes and cytokines elicited by Dectin-2 cross-linking, confirming the importance of this molecule in Dectin-2 signaling. Using an adoptive transfer model, we demonstrate that inhibition of PI3Kδ profoundly reduces the capacity of bone marrow-derived dendritic cells to sensitize recipient mice for Th2 and Th17 pulmonary inflammation in response to D. farinae Furthermore, administration of a PI3Kδ inhibitor during the sensitization of wild-type mice prevents the generation of D. farinae-induced pulmonary inflammation. These results demonstrate that PI3Kδ regulates Dectin-2 signaling and its dendritic cell function. PMID:27194783

  18. Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

    Directory of Open Access Journals (Sweden)

    Olsen Birgitte B

    2012-03-01

    Full Text Available Abstract Background The DNA-dependent protein kinase (DNA-PK is a nuclear complex composed of a large catalytic subunit (DNA-PKcs and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ repair mechanism, which is activated in the presence of DNA double-strand breaks induced by ionizing radiation, reactive oxygen species and radiomimetic drugs. We have recently reported that down-regulation of protein kinase CK2 by siRNA interference results in enhanced cell death specifically in DNA-PKcs-proficient human glioblastoma cells, and this event is accompanied by decreased autophosphorylation of DNA-PKcs at S2056 and delayed repair of DNA double-strand breaks. Results In the present study, we show that CK2 co-localizes with phosphorylated histone H2AX to sites of DNA damage and while CK2 gene knockdown is associated with delayed DNA damage repair, its overexpression accelerates this process. We report for the first time evidence that lack of CK2 destabilizes the interaction of DNA-PKcs with DNA and with Ku80 at sites of genetic lesions. Furthermore, we show that CK2 regulates the phosphorylation levels of DNA-PKcs only in response to direct induction of DNA double-strand breaks. Conclusions Taken together, these results strongly indicate that CK2 plays a prominent role in NHEJ by facilitating and/or stabilizing the binding of DNA-PKcs and, possibly other repair proteins, to the DNA ends contributing to efficient DNA damage repair in mammalian cells.

  19. Differential Roles of Two Homologous Cyclin-Dependent Kinase Inhibitor Genes in Regulating Cell Cycle and Innate Immunity in Arabidopsis.

    Science.gov (United States)

    Hamdoun, Safae; Zhang, Chong; Gill, Manroop; Kumar, Narender; Churchman, Michelle; Larkin, John C; Kwon, Ashley; Lu, Hua

    2016-01-01

    Precise cell-cycle control is critical for plant development and responses to pathogen invasion. Two homologous cyclin-dependent kinase inhibitor genes, SIAMESE (SIM) and SIM-RELATED 1 (SMR1), were recently shown to regulate Arabidopsis (Arabidopsis thaliana) defense based on phenotypes conferred by a sim smr1 double mutant. However, whether these two genes play differential roles in cell-cycle and defense control is unknown. In this report, we show that while acting synergistically to promote endoreplication, SIM and SMR1 play different roles in affecting the ploidy of trichome and leaf cells, respectively. In addition, we found that the smr1-1 mutant, but not sim-1, was more susceptible to a virulent Pseudomonas syringae strain, and this susceptibility could be rescued by activating salicylic acid (SA)-mediated defense. Consistent with these results, smr1-1 partially suppressed the dwarfism, high SA levels, and cell death phenotypes in acd6-1, a mutant used to gauge the change of defense levels. Thus, SMR1 functions partly through SA in defense control. The differential roles of SIM and SMR1 are due to differences in temporal and spatial expression of these two genes in Arabidopsis tissues and in response to P. syringae infection. In addition, flow-cytometry analysis of plants with altered SA signaling revealed that SA is necessary, but not sufficient, to change cell-cycle progression. We further found that a mutant with three CYCD3 genes disrupted also compromised disease resistance to P. syringae. Together, this study reveals differential roles of two homologous cyclin-dependent kinase inhibitors in regulating cell-cycle progression and innate immunity in Arabidopsis and provides insights into the importance of cell-cycle control during host-pathogen interactions. PMID:26561564

  20. Role of calcium and AMP kinase in the regulation of mitochondrial biogenesis and GLUT4 levels in muscle.

    Science.gov (United States)

    Ojuka, Edward O

    2004-05-01

    Contractile activity induces mitochondrial biogenesis and increases glucose transport capacity in muscle. There has been much research on the mechanisms responsible for these adaptations. The present paper reviews the evidence, which indicates that the decrease in the levels of high-energy phosphates, leading to activation of AMP kinase (AMPK), and the increase in cytosolic Ca(2+), which activates Ca(2+)/calmodulin-dependent protein kinase (CAMK), are signals that initiate these adaptative responses. Although the events downstream of AMPK and CAMK have not been well characterized, these events lead to activation of various transcription factors, including: nuclear respiratory factors (NRF) 1 and 2, which cause increased expression of proteins of the respiratory chain; PPAR-alpha, which up regulates the levels of enzymes of beta oxidation; mitochondrial transcription factor A, which activates expression of the mitochondrial genome; myocyte-enhancing factor 2A, the transcription factor that regulates GLUT4 expression. The well-orchestrated expression of the multitude of proteins involved in these adaptations is mediated by the rapid activation of PPAR gamma co-activator (PGC) 1, a protein that binds to various transcription factors to maximize transcriptional activity. Activating AMPK using 5-aminoimidizole-4-carboxamide-1-beta-D-riboside (AICAR) and increasing cytoplasmic Ca(2+) using caffeine, W7 or ionomycin in L6 myotubes increases the concentration of mitochondrial enzymes and GLUT4 and enhances the binding of NRF-1 and NRF-2 to DNA. AICAR and Ca-releasing agents also increase the levels of PGC-1, mitochondrial transcription factor A and myocyte-enhancing factors 2A and 2D. These results are similar to the responses seen in muscle during the adaptation to endurance exercise and show that L6 myotubes are a suitable model for studying the mechanisms by which exercise causes the adaptive responses in muscle mitochondria and glucose transport. PMID:15294043

  1. Extracellular signal-regulated kinase 2 (ERK2) phosphorylation sites and docking domain on the nuclear pore complex protein Tpr cooperatively regulate ERK2-Tpr interaction.

    Science.gov (United States)

    Vomastek, Tomás; Iwanicki, Marcin P; Burack, W Richard; Tiwari, Divya; Kumar, Devanand; Parsons, J Thomas; Weber, Michael J; Nandicoori, Vinay Kumar

    2008-11-01

    Identifying direct substrates of mitogen-activated protein kinases (MAPKs) and understanding how those substrates are selected is central to understanding how these ubiquitously activated enzymes generate diverse biological responses. In previous work, we identified several new candidate substrates for the MAPK ERK2 (extracellular signal-regulated kinase 2), including the nuclear pore complex protein Tpr (translocated promoter region). In this report, we identify sites on Tpr for ERK2 phosphorylation and binding and demonstrate their functional interaction. ERK2 phosphorylation and dimerization are necessary for ERK2-Tpr binding, and this occurs through a DEF (docking site for ERK2, FXF) domain on Tpr. Surprisingly, the DEF domain and the phosphorylation sites displayed positive cooperativity to promote ERK2 binding to Tpr, in contrast to substrates where phosphorylation reduces binding. Ectopic expression or depletion of Tpr resulted in decreased movement of activated ERK2 from the cytoplasm to the nucleus, implying a role for Tpr in ERK2 translocation. Collectively, the data provide direct evidence that a component of the nuclear pore complex is a bona fide substrate of ERK2 in vivo and that activated ERK2 stably associates with this substrate after phosphorylation, where it could play a continuing role in nuclear pore function. We propose that Tpr is both a substrate and a scaffold for activated ERKs.

  2. Activated Integrin-Linked Kinase Negatively Regulates Muscle Cell Enhancement Factor 2C in C2C12 Cells

    Directory of Open Access Journals (Sweden)

    Zhenguo Dong

    2015-01-01

    Full Text Available Our previous study reported that muscle cell enhancement factor 2C (MEF2C was fully activated after inhibition of the phosphorylation activity of integrin-linked kinase (ILK in the skeletal muscle cells of goats. It enhanced the binding of promoter or enhancer of transcription factor related to proliferation of muscle cells and then regulated the expression of these genes. In the present investigation, we explored whether ILK activation depended on PI3K to regulate the phosphorylation and transcriptional activity of MEF2C during C2C12 cell proliferation. We inhibited PI3K activity in C2C12 with LY294002 and then found that ILK phosphorylation levels and MEF2C phosphorylation were decreased and that MCK mRNA expression was suppressed significantly. After inhibiting ILK phosphorylation activity with Cpd22 and ILK-shRNA, we found MEF2C phosphorylation activity and MCK mRNA expression were increased extremely significantly. In the presence of Cpd22, PI3K activity inhibition increased MEF2C phosphorylation and MCK mRNA expression indistinctively. We conclude that ILK negatively and independently of PI3K regulated MEF2C phosphorylation activity and MCK mRNA expression in C2C12 cells. The results provide new ideas for the study of classical signaling pathway of PI3K-ILK-related proteins and transcription factors.

  3. Aurora A Kinase Regulates Mammary Epithelial Cell Fate by Determining Mitotic Spindle Orientation in a Notch-Dependent Manner

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    Joseph L. Regan

    2013-07-01

    Full Text Available Cell fate determination in the progeny of mammary epithelial stem/progenitor cells remains poorly understood. Here, we have examined the role of the mitotic kinase Aurora A (AURKA in regulating the balance between basal and luminal mammary lineages. We find that AURKA is highly expressed in basal stem cells and, to a lesser extent, in luminal progenitors. Wild-type AURKA expression promoted luminal cell fate, but expression of an S155R mutant reduced proliferation, promoted basal fate, and inhibited serial transplantation. The mechanism involved regulation of mitotic spindle orientation by AURKA and the positioning of daughter cells after division. Remarkably, this was NOTCH dependent, as NOTCH inhibitor blocked the effect of wild-type AURKA expression on spindle orientation and instead mimicked the effect of the S155R mutant. These findings directly link AURKA, NOTCH signaling, and mitotic spindle orientation and suggest a mechanism for regulating the balance between luminal and basal lineages in the mammary gland.

  4. Regulation of salt and ABA responses by CIPK14, a calcium sensor interacting protein kinase in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    QIN YuZhi; LI Xu; GUO Ming; DENG KeQin; LIN dianZhong; TANG DongYing; GUO XinHong; LIU XuanMing

    2008-01-01

    Calcium and protein kinsse serve as the common mediators to regulate plant responses to multiple stresses including salt and ABA stimulus. Here we reported a novel protein kinase (CIPK14) that regulated the responses to ABA treatment and salt stress in Arabidopsis. CIPK14 transcripts, capable been checked in roots, stems, leaves and flowers, were highly expressed in flowers and roots. CIPK14 was induced by ABA and salt treatments. The disruption of CIPK14 altered the transcriptional pattern of a gene marker line related to ABA and salt responses, and the results suggested that CIPK14 probably was responsible to the control of the salt and ABA responses. Comparing with wild types, the lines inserted with the T-DNA in which CIPK14 gene expression was knocked out were also more sensitive to ABA and salt stimulus, showing low germination rate and the less root elongation. While, when these conditioned seeds were treated with norflurazon, their germination percentages could recover to a certain extent. We also found that exogenous calcium could have an effect on the transcription of CIPK14 under ABA end salt treatments, and it seemed that calcium ion might work upstream ClPK14 to regulate the plant response to ABA and salt response.

  5. Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

    Science.gov (United States)

    Lu, Y. T.; Hidaka, H.; Feldman, L. J.

    1996-01-01

    Roots of many species respond to gravity (gravitropism) and grow downward only if illuminated. This light-regulated root gravitropism is phytochrome-dependent, mediated by calcium, and inhibited by KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II (CaMK II). A cDNA encoding MCK1, a maize homolog of mammalian CaMK, has been isolated from roots of maize (Zea mays L.). The MCK1 gene is expressed in root tips, the site of perception for both light and gravity. Using the [35S]CaM gel-overlay assay we showed that calmodulin-binding activity of the MCK1 is abolished by 50 microM KN-93, but binding is not affected by 5 microM KN-93, paralleling physiological findings that light-regulated root gravitropism is inhibited by 50 microM KN-93, but not by 5 microM KN-93. KN-93 inhibits light-regulated gravitropism by interrupting transduction of the light signal, not light perception, suggesting that MCK1 may play a role in transducing light. This is the first report suggesting a physiological function for a CaMK homolog in light signal transduction.

  6. Epidermal Growth Factor Stimulates Extracellular-Signal Regulated Kinase Phosphorylation of a Novel Site on Cytoplasmic Dynein Intermediate Chain 2

    Directory of Open Access Journals (Sweden)

    Andrew D. Catling

    2013-02-01

    Full Text Available Extracellular-signal regulated kinase (ERK signaling is required for a multitude of physiological and patho-physiological processes. However, the identities of the proteins that ERK phosphorylates to elicit these responses are incompletely known. Using an affinity purification methodology of general utility, here we identify cytoplasmic dynein intermediate chain 2 (DYNC1I-2, IC-2 as a novel substrate for ERK following epidermal growth factor receptor stimulation of fibroblasts. IC-2 is a subunit of cytoplasmic dynein, a minus-end directed motor protein necessary for transport of diverse cargos along microtubules. Emerging data support the hypothesis that post-translational modification regulates dynein but the signaling mechanisms used are currently unknown. We find that ERK phosphorylates IC-2 on a novel, highly conserved Serine residue proximal to the binding site for the p150Glued subunit of the cargo adapter dynactin. Surprisingly, neither constitutive phosphorylation nor a phosphomimetic substitution of this Serine influences binding of p150Glued to IC-2. These data suggest that ERK phosphorylation of IC-2 regulates dynein function through mechanisms other than its interaction with dynactin.

  7. The Sensor Kinase GacS Negatively Regulates Flagellar Formation and Motility in a Biocontrol Bacterium, Pseudomonas chlororaphis O6

    Directory of Open Access Journals (Sweden)

    Ji Soo Kim

    2014-06-01

    Full Text Available The GacS/GacA two component system regulates various traits related to the biocontrol potential of plant-associated pseudomonads. The role of the sensor kinase, GacS, differs between strains in regulation of motility. In this study, we determined how a gacS mutation changed cell morphology and motility in Pseudomonas chlororaphis O6. The gacS mutant cells were elongated in stationary-phase compared to the wild type and the complemented gacS mutant, but cells did not differ in length in logarithmic phase. The gacS mutant had a two-fold increase in the number of flagella compared with the wild type strain; flagella number was restored to that of the wild type in the complemented gacS mutant. The more highly flagellated gacS mutant cells had greater swimming motilities than that of the wild type strain. Enhanced flagella formation in the gacS mutant correlated with increased expression of three genes, fleQ, fliQ and flhF, involved in flagellar formation. Expression of these genes in the complemented gacS mutant was similar to that of the wild type. These findings show that this root-colonizing pseudomonad adjusts flagella formation and cell morphology in stationary-phase using GacS as a major regulator.

  8. Expression of a TGF-{beta} regulated cyclin-dependent kinase inhibitor in normal and immortalized airway epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Tierney, L.A.; Bloomfield, C.; Johnson, N.F. [and others

    1995-12-01

    Tumors arising from epithelial cells, including lung cancers are frequently resistant to factors that regulate growth and differentiation in normal in normal cells. Once such factor is transforming growth factor-{Beta} (TGF-{Beta}). Escape from the growth-inhibitory effects of TGF-{Beta} is thought to be a key step in the transformation of airway epithelial cells. most lung cancer cell lines require serum for growth. In contrast, normal human bronchial epithelial (NHBE) cells are exquisitely sensitive to growth-inhibitory and differentiating effects of TGF-{Beta}. The recent identification of a novel cyclin-dependent kinase inhibitor, p15{sup INK4B}, which is regulated by TGF-{Beta}, suggests a mechanism by which TGF-{Beta} mediates growth arrest in NHBE cells. The purpose of this study was two-fold: (1) to determine if p15{sup INK4B} is induced by TGF-{Beta} in NHBE cells or immortalized bronchial epithelial (R.1) cells and if that induction corresponds to a G1/S cell-cycle arrest; (2) to determine the temporal relationship between p15{sup INK4B} induction, cell-cycle arrest, and the phosphorylation state of the pRB because it is thought that p15{sup INK4B} acts indirectly by preventing phosphorylation of the RB gene product. In this study, expression of p15{sup INK4B} was examined in NHBE cells and R.1 cells at different time intervals following TGF-{Beta} treatment. The expression of this kinase inhibitor and its relationship to the cell and the pRb phosphorylation state were examined in cells that were both sensitive (NHBE) and resistant (R.1) to the effects of TGF-{Beta}. These results suggest that the cyclin-dependent kinase inhibitor, p15{sup INK4B}, is involved in airway epithelial cell differentiation and that loss or reduction of expression plays a role in the resistance of transformed or neoplastic cells to the growth-inhibitory effects of TGF-{Beta}.

  9. AMP-activated protein kinase (AMPK mediates nutrient regulation of thioredoxin-interacting protein (TXNIP in pancreatic beta-cells.

    Directory of Open Access Journals (Sweden)

    Maayan Shaked

    Full Text Available Thioredoxin-interacting protein (TXNIP regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP. Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment

  10. Glutathione S-transferase class mu regulation of apoptosis signal-regulating kinase 1 protein during VCD-induced ovotoxicity in neonatal rat ovaries

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharya, Poulomi; Madden, Jill A. [Department of Animal Science, Iowa State University, Ames, IA 50011 (United States); Sen, Nivedita; Hoyer, Patricia B. [Department of Physiology, University of Arizona, Tucson, AZ 85724 (United States); Keating, Aileen F., E-mail: akeating@iastate.edu [Department of Animal Science, Iowa State University, Ames, IA 50011 (United States)

    2013-02-15

    4-Vinylcyclohexene diepoxide (VCD) destroys ovarian primordial and small primary follicles via apoptosis. In mice, VCD exposure induces ovarian mRNA expression of glutathione S-transferase (GST) family members, including isoform mu (Gstm). Extra-ovarian GSTM negatively regulates pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1) through protein complex formation, which dissociates during stress, thereby initiating ASK1-induced apoptosis. The present study investigated the ovarian response of Gstm mRNA and protein to VCD. Induction of Ask1 mRNA at VCD-induced follicle loss onset was determined. Ovarian GSTM:ASK1 protein complex formation was investigated and VCD exposure effects thereon evaluated. Phosphatidylinositol-3 kinase (PI3K) regulation of GSTM protein was also studied. Postnatal day (PND) 4 rat ovaries were cultured in control media ± 1) VCD (30 μM) for 2–8 days; 2) VCD (30 μM) for 2 days, followed by incubation in control media for 4 days (acute VCD exposure); or 3) LY294002 (20 μM) for 6 days. VCD exposure did not alter Gstm mRNA expression, however, GSTM protein increased (P < 0.05) after 6 days of both the acute and chronic treatments. Ask1 mRNA increased (0.33-fold; P < 0.05) relative to control after 6 days of VCD exposure. Ovarian GSTM:ASK1 protein complex formation was confirmed and, relative to control, the amount of GSTM bound to ASK1 increased 33% (P < 0.05) by chronic but with no effect of acute VCD exposure. PI3K inhibition increased (P < 0.05) GSTM protein by 40% and 71% on d4 and d6, respectively. These findings support involvement of GSTM in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1. - Highlights: ► GSTM protein increases in response to ovarian VCD exposure. ► VCD increases Ask1 mRNA at the onset of follicle loss. ► Ovarian GSTM binds more ASK1 protein during VCD-induced ovotoxicity. ► PI3K regulates ovarian GSTM protein.

  11. Regulation of skeletal muscle sucrose, non-fermenting 1/AMP-activated protein kinase-related kinase (SNARK) by metabolic stress and diabetes.

    OpenAIRE

    Rune, A.; Osler, M. E.; Fritz, T.; Zierath, J. R.

    2010-01-01

    Aims/hypothesis Sucrose, non-fermenting 1/AMP-activated protein kinase-related kinase (SNARK) is involved in cellular stress responses linked to obesity and type 2 diabetes. We determined the role of SNARK in response to metabolic stress and insulin action on glucose and lipid metabolism in skeletal muscle. Methods Vastus lateralis skeletal muscle biopsies were obtained from normal glucose tolerant (n = 35) and type 2 diabetic (n = 31) men and women for SNARK expression studies. Primary myotu...

  12. A-RAF kinase functions in ARF6 regulated endocytic membrane traffic.

    Directory of Open Access Journals (Sweden)

    Elena Nekhoroshkova

    Full Text Available BACKGROUND: RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation. METHODOLOGY/PRINCIPAL FINDINGS: Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149 that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment. CONCLUSIONS/SIGNIFICANCE: A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking.

  13. Danthron activates AMP-activated protein kinase and regulates lipid and glucose metabolism in vitro

    Institute of Scientific and Technical Information of China (English)

    Rong ZHOU; Ling WANG; Xing XU; Jing CHEN; Li-hong HU; Li-li CHEN; Xu SHEN

    2013-01-01

    Aim:To discover the active compound on AMP-activated protein kinase (AMPK) activation and investigate the effects of the active compound 1,8-dihydroxyanthraquinone (danthron) from the traditional Chinese medicine rhubarb on AMPK-mediated lipid and glucose metabolism in vitro.Methods:HepG2 and C2C12 cells were used.Cell viability was determined using MTT assay.Real-time PCR was performed to measure the gene expression.Western blotting assay was applied to investigate the protein phosphorylation level.Enzymatic assay kits were used to detect the total cholesterol (TC),triglyceride (TG) and glucose contents.Results:Danthron (0.1,1,and 10 μmol/L) dose-dependently promoted the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC)in both HepG2 and C2C12 cells.Meanwhile,danthron treatment significantly reduced the lipid synthesis related sterol regulatory element-binding protein 1c (SREBP1c) and fatty acid synthetase (FAS) gene expressions,and the TC and TG levels.In addition,danthron treatment efficiently increased glucose consumption.The actions of danthron on lipid and glucose metabolism were abolished or reversed by co-treatment with the AMPK inhibitor compound C.Conclusion:Danthron effectively reduces intracellular lipid contents and enhanced glucose consumption in vitro via activation of AMPK signaling pathway.

  14. Dynamin forms a Src kinase-sensitive complex with Cbl and regulates podosomes and osteoclast activity.

    Science.gov (United States)

    Bruzzaniti, Angela; Neff, Lynn; Sanjay, Archana; Horne, William C; De Camilli, Pietro; Baron, Roland

    2005-07-01

    Podosomes are highly dynamic actin-containing adhesion structures found in osteoclasts, macrophages, and Rous sarcoma virus (RSV)-transformed fibroblasts. After integrin engagement, Pyk2 recruits Src and the adaptor protein Cbl, forming a molecular signaling complex that is critical for cell migration, and deletion of any molecule in this complex disrupts podosome ring formation and/or decreases osteoclast migration. Dynamin, a GTPase essential for endocytosis, is also involved in actin cytoskeleton remodeling and is localized to podosomes where it has a role in actin turnover. We found that dynamin colocalizes with Cbl in the actin-rich podosome belt of osteoclasts and that dynamin forms a complex with Cbl in osteoclasts and when overexpressed in 293VnR or SYF cells. The association of dynamin with Cbl in osteoclasts was decreased by Src tyrosine kinase activity and we found that destabilization of the dynamin-Cbl complex involves the recruitment of Src through the proline-rich domain of Cbl. Overexpression of dynamin increased osteoclast bone resorbing activity and migration, whereas overexpression of dynK44A decreased osteoclast resorption and migration. These studies suggest that dynamin, Cbl, and Src coordinately participate in signaling complexes that are important in the assembly and remodeling of the actin cytoskeleton, leading to changes in osteoclast adhesion, migration, and resorption. PMID:15872089

  15. Elastic network model of allosteric regulation in protein kinase PDK1

    Directory of Open Access Journals (Sweden)

    Williams Gareth

    2010-05-01

    Full Text Available Abstract Background Structural switches upon binding of phosphorylated moieties underpin many signalling networks. The ligand activation is a form of allosteric modulation of the protein, where the binding site is remote from the structural change in the protein. Recently this structural switch has been elegantly demonstrated with the crystallisation of the activated form of 3-phosphoinositide-dependent protein kinase-1 (PDK1. The purpose of the present work is to determine whether the allosteric coupling in PDK1 emerges at the level of a simple coarse grained model of protein dynamics. Results It is shown here that the allosteric effects of the agonist binding to the small lobe upon the activation loop in the large lobe of PDK1 are explainable within a simple 'ball and spring' elastic network model (ENM of protein dynamics. In particular, the model shows that the bound phospho peptide mimetic fluctuations have a high degree of correlation with the activation loop of PDK1. Conclusions The ENM approach to small molecule activation of proteins may offer a first pass predictive methodology where affinity is encoded in residues remote from the active site, and aid in the design of specific protein agonists that enhance the allosteric coupling and antagonist that repress it.

  16. The tyrosine kinase Btk regulates the macrophage response to Listeria monocytogenes infection.

    Directory of Open Access Journals (Sweden)

    Afitap Derya Köprülü

    Full Text Available In this study we investigated the role of Bruton's tyrosine kinase (Btk in the immune response to the Gram-positive intracellular bacterium Listeria monocytogenes (Lm. In response to Lm infection, Btk was activated in bone marrow-derived macrophages (BMMs and Btk (-/- BMMs showed enhanced TNF-α, IL-6 and IL-12p40 secretion, while type I interferons were produced at levels similar to wild-type (wt BMMs. Although Btk-deficient BMMs displayed reduced phagocytosis of E. coli fragments, there was no difference between wt and Btk (-/- BMMs in the uptake of Lm upon infection. Moreover, there was no difference in the response to heat-killed Lm between wt and Btk (-/- BMMs, suggesting a role for Btk in signaling pathways that are induced by intracellular Lm. Finally, Btk (-/- mice displayed enhanced resistance and an increased mean survival time upon Lm infection in comparison to wt mice. This correlated with elevated IFN-γ and IL-12p70 serum levels in Btk (-/- mice at day 1 after infection. Taken together, our data suggest an important regulatory role for Btk in macrophages during Lm infection.

  17. [Receptor tyrosine kinase KIT may regulate expression of genes involved in spontaneous regression of neuroblastoma].

    Science.gov (United States)

    Lebedev, T D; Spirin, P V; Suntsova, M V; Ivanova, A V; Buzdin, A A; Prokofjeva, M M; Rubtsov, P M; Prassolov, V S

    2015-01-01

    Hallmark of neuroblastoma is an ability of this malignant tumor to undergo spontaneous regression or differentiation into benign tumor during any stage of the disease, but it is little known about mechanisms of these phenomena. We studied effect of receptor tyrosine kinase receptor KIT on expression of genes, which may be involved in tumor spontaneous regression. Downregulation of KIT expression by RNA interference in SH-SY5Y cells causes suppression of neurotrophin receptor NGFR expression that may promote the loss of sensibility of cells to nerve growth factors, also it causes upregulation of TrkA receptor expression which can stimulate cell differentiation or apoptosis in NGF dependent manner. Furthermore there is an upregulation of genes which stimulate malignant cell detection by immune system, such as genes of major histocompatibility complex HLA class I HLA-B and HLA-C, and interferon-γ receptors IFNGR1 and IFNGR2 genes. Thus KIT can mediate neuroblastoma cell sensibility to neurotrophins and immune system components--two factors directly contributing to spontaneous regression of neuroblastoma.

  18. Cutting Edge: RIP1 kinase activity is dispensable for normal development but is a key regulator of inflammation in SHARPIN-deficient mice.

    Science.gov (United States)

    Berger, Scott B; Kasparcova, Viera; Hoffman, Sandy; Swift, Barb; Dare, Lauren; Schaeffer, Michelle; Capriotti, Carol; Cook, Michael; Finger, Joshua; Hughes-Earle, Angela; Harris, Philip A; Kaiser, William J; Mocarski, Edward S; Bertin, John; Gough, Peter J

    2014-06-15

    RIP1 (RIPK1) kinase is a key regulator of TNF-induced NF-κB activation, apoptosis, and necroptosis through its kinase and scaffolding activities. Dissecting the balance of RIP1 kinase activity and scaffolding function in vivo during development and TNF-dependent inflammation has been hampered by the perinatal lethality of RIP1-deficient mice. In this study, we generated RIP1 kinase-dead (Ripk1(K45A)) mice and showed they are viable and healthy, indicating that the kinase activity of RIP1, but not its scaffolding function, is dispensable for viability and homeostasis. After validating that the Ripk1(K45A) mice were specifically protected against necroptotic stimuli in vitro and in vivo, we crossed them with SHARPIN-deficient cpdm mice, which develop severe skin and multiorgan inflammation that has been hypothesized to be mediated by TNF-dependent apoptosis and/or necroptosis. Remarkably, crossing Ripk1(K45A) mice with the cpdm strain protected against all cpdm-related pathology. Together, these data suggest that RIP1 kinase represents an attractive therapeutic target for TNF-driven inflammatory diseases.

  19. cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta

    NARCIS (Netherlands)

    A.B. Vaandrager (Arie); S.M. Lohmann (Suzanne); H.R. de Jonge (Hugo); W.C. Poller; B.C. Tilly (Bernard); A. Smolenski; S. Schneider-Rasp; A.G. Bot (Alice); M.J. Edixhoven (Marcel); B.J. Scholte (Bob); T. Jarchau; U. Walter

    1997-01-01

    textabstractIn order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was ana

  20. Isotype-specific activation of cystic fibrosis transmembrane conductance regulator-chloride channels by cGMP-dependent protein kinase II

    NARCIS (Netherlands)

    P.J. French (Pim); J. Bijman (Jan); M.J. Edixhoven (Marcel); A.B. Vaandrager (Arie); B.J. Scholte (Bob); S.M. Lohmann (Suzanne); A.C. Nairn; H.R. de Jonge (Hugo)

    1995-01-01

    textabstractType II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type I alpha cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl- channel in excis

  1. Metaplasticity gated through differential regulation of GluN2A versus GluN2B receptors by Src family kinases

    OpenAIRE

    Yang, Kai; Trepanier, Catherine; Sidhu, Bikram; Xie, Yu-Feng; Li, Hongbin; Lei, Gang; Michael W. Salter; Orser, Beverley A.; Nakazawa, Takanobu; Yamamoto, Tadashi; Jackson, Michael F.; MacDonald, John F.

    2011-01-01

    Synaptic plasticity, which forms the basis of learning and memory, is controlled by GPCRs. GPCR signalling results in either long-term potentiation (LTP) or depression (LTD) by differentially regulating NMDA receptor subtypes via activation of distinct Src kinase family members.

  2. Increased limbic phosphorylated extracellular-regulated kinase 1 and 2 expression after chronic stress is reduced by cyclic 17 beta-estradiol administration

    NARCIS (Netherlands)

    Gerrits, M.; Westenbroek, C.; Koch, T.; Grootkarzijn, A.; Ter Horst, G. J.

    2006-01-01

    Chronic stress induced neuronal changes that may have consequences for subsequent stress responses. For example, chronic stress in rats rearranges dendritic branching patterns and disturbs the phosphorylation of extracellular-regulated kinase 1 and 2 (ERK) 1/2 throughout the limbic system. Stress-in

  3. Receptor tyrosine kinase-like orphan receptor 1, a target of NKX2-1/TTF-1 lineage-survival oncogene, inhibits apoptosis signal-regulating kinase 1-mediated pro-apoptotic signaling in lung adenocarcinoma.

    Science.gov (United States)

    Ida, Lisa; Yamaguchi, Tomoya; Yanagisawa, Kiyoshi; Kajino, Taisuke; Shimada, Yukako; Suzuki, Motoshi; Takahashi, Takashi

    2016-02-01

    We previously identified receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a transcriptional target of the NKX2-1/TTF-1 lineage-survival oncogene in lung adenocarcinoma. ROR1 consequently sustains a favorable balance between pro-survival phosphatidylinositol 3-kinase-protein kinase B and pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1)-p38MAPK signaling. In contrast to recent advances in understanding how ROR1 sustains pro-survival signaling, the mechanism of ROR1 repression of pro-apoptotic signaling remains rather elusive. In the present study, we investigated the underlying mechanism of ROR1-mediated inhibition of the ASK1-p38MAPK signaling pathway. Growth inhibition mediated by siROR1 was partially but significantly alleviated by ASK1 co-knockdown in lung adenocarcinoma cell lines. Also, ASK1 phosphorylation at Thr845, which reflects its activated state, was clearly inhibited by ROR1 overexpression in both steady state and oxidative stress-elicited conditions in MSTO-211H cells. In addition, we found that ROR1 was physically associated with ASK1 at the C-terminal serine threonine-rich domain of ROR1. Furthermore, ROR1 kinase activity was shown to be required to repress the ASK1-p38 axis and oxidative stress-induced cell death. The present findings thus support our notion that ROR1 sustains lung adenocarcinoma survival, at least in part, through direct physical interaction with ASK1 and consequential repression of the pro-apoptotic ASK1-p38 axis in a ROR1 kinase activity-dependent manner. PMID:26661061

  4. Serine/threonine kinase-protein kinase B and extracellular signal-regulated kinase regulate ventilator-induced pulmonary fibrosis after bleomycin-induced acute lung injury: a prospective, controlled animal experiment

    OpenAIRE

    Li, Li-Fu; Liao, Shuen-Kuei; Huang, Chung-Chi; Hung, Ming-Jui; Quinn, Deborah A

    2008-01-01

    Introduction Lung fibrosis, reduced lung compliance, and severe hypoxemia found in patients with acute lung injury often result in a need for the support of mechanical ventilation. High-tidal-volume mechanical ventilation can increase lung damage and fibrogeneic activity but the mechanisms regulating the interaction between high tidal volume and lung fibrosis are unclear. We hypothesized that high-tidal-volume ventilation increased pulmonary fibrosis in acute lung injury via the serine/threon...

  5. miR-155-dependent regulation of mammalian sterile 20-like kinase 2 (MST2) coordinates inflammation, oxidative stress and proliferation in vascular smooth muscle cells.

    Science.gov (United States)

    Yang, Zhan; Zheng, Bin; Zhang, Yu; He, Ming; Zhang, Xin-hua; Ma, Dong; Zhang, Ruo-nan; Wu, Xiao-li; Wen, Jin-kun

    2015-07-01

    In response to vascular injury, inflammation, oxidative stress, and cell proliferation often occur simultaneously in vascular tissues. We previously observed that microRNA-155 (miR-155), which is implicated in proliferation and inflammation is involved in neointimal hyperplasia; however, the molecular mechanisms by which it regulates these processes remain largely unknown. In this study, we observed that vascular smooth muscle cell (VSMC) proliferation and neointimal formation in wire-injured femoral arteries were reduced by the loss of miR-155 and increased by the gain of miR-155. The proliferative effect of miR-155 was also observed in cultured VSMCs. Notably, expression of the miR-155-target protein mammalian sterile 20-like kinase 2 (MST2) was increased in the injured arteries of miR-155-/- mice. miR-155 directly repressed MST2 and thus activated the extracellular signal-regulated kinase (ERK) pathway by promoting an interaction between RAF proto-oncogene serine/threonine-protein kinase (Raf-1) and mitogen-activated protein kinase kinase (MEK) and stimulating inflammatory and oxidative stress responses; together, these effects lead to VSMC proliferation and vascular remodeling. Our data reveal that MST2 mediates miR-155-promoted inflammatory and oxidative stress responses by altering the interaction of MEK with Raf-1 and MST2 in response to vascular injury. Therefore, suppression of endogenous miR-155 might be a novel therapeutic strategy for vascular injury and remodeling. PMID:25892184

  6. An experimental study of extracellular signal-regulated kinase and its inter ventional treatments in hepatic ifbrosis

    Institute of Scientific and Technical Information of China (English)

    Ming-De Jiang; Shu-Mei Zheng; Hui Xu; Wei-Zheng Zeng; Yong Zhang; Hao-Ping Sun; Yun-Xia Wang; Jian-Ping Qin; Xiao-Ling Wu

    2008-01-01

    BACKGROUND:The pathogenesis of hepatic ifbrosis and cirrhosis is still not fully understood. The extracellular signal-regulated kinase (ERK) pathway is involved in the regulation of cell proliferation and differentiation. The aim of this study was to investigate the effects of PD98059, a speciifc inhibitor of ERK, on the cell cycle, cell proliferation, secretion of typeⅠcollagen and expression of cyclin D1 mRNA, CDK4 mRNA and transforming growth factor-β1 (TGF-β1) mRNA in rat hepatic stellate cells (HSCs) stimulated by acetaldehyde. METHODS:Rat HSCs stimulated by acetaldehyde were incubated with PD98059 at different concentrations. The cell cycle was analysed by lfow cytometry. Cell proliferation was assessed by the methyl thiazolyl tetrazolium colorimetric assay. The mRNA expression of cyclin D1, CDK4 and TGF-β1 was examined using the reverse transcriptase-polymerase chain reaction. Type Ⅰcollagen in the culture medium was detected by enzyme-linked immunosorbent assay. RESULTS: 20, 50 and 100 μmol/L PD98059 signiifcantly inhibited the proliferation and provoked a G0/G1-phase arrest of acetaldehyde-induced HSCs in a dose-dependent manner. The secretion of typeⅠ collagen and the expression of cyclin D1, CDK4 and TGF-β1 mRNA in acetaldehyde-induced HSCs were markedly inhibited by 50 and 100μmol/L PD98059, respectively. CONCLUSIONS:The ERK pathway regulates the cell proliferation, secretion of type Ⅰ collagen and the expression of TGF-β1 mRNA in rat HSCs stimulated by acetaldehyde, which is likely related to its regulative effect on the cell cycle.

  7. Leptin Regulated Insulin Secretion via Stimulating IRS2-associated Phosphoinositide 3-kinase Activity in the isolated Rat Pancreatic Islets

    Institute of Scientific and Technical Information of China (English)

    袁莉; 安汉祥; 李卓娅; 邓秀玲

    2003-01-01

    To investigate the molecular mechanism of leptin regulating insulin secretion through determining the regulation of insulin secretion and the insulin receptor substrate (IRS)-2-associated phosphoinositide 3-kinase (PI3K) activity by leptin in the isolated rat pancreatic islets, pancreatic islets were isolated from male SD rats by the collagenase method. The purified islets were incubated with leptin 2 nmol/L for 1 h in the presence of 5.6 mmol/L or 11.1 mmol/L glucose. Insulin release was measured using radioimmunoassay. IRS-2-associated activity of PI3K was determined by immunoprecipitate assay and Western blot. The results showed that in the presence of 5.6 mmol/L glucose, leptin had no significant effect on both insulin secretion and IRS-2-associated PI3K activity, but in the presence of 11.1 mmol/L glucose, insulin release was significantly inhibited after the islets were exposed to leptin for 1 h (P<0. 01). PI3K inhibitor wortmannin blocked the inhibitory regulation of leptin on insulin release (P<0. 05). Western Blot assay revealed that 2 nmol/L leptin could significantly increase the IRS-2-associated activity of PI3K by 51.5 % (P<0. 05) in the presence of 11.1 mmol/L glucose. It was concluded that Leptin could significantly inhibit insulin secretion in the presence of 11.1 mmol/L glucose by stimulating IRS-2-associated activity of PI3K, which might be the molecular mechanism of leptin regulating insulin secretion.

  8. Regulators of cyclin-dependent kinases are crucial for maintaining genome integrity in S phase

    DEFF Research Database (Denmark)

    Beck, Halfdan; Nähse, Viola; Larsen, Marie Sofie Yoo;

    2010-01-01

    are important negative regulators of CDK1 and -2. Strikingly, WEE1 depletion rapidly induced DNA damage in S phase in newly replicated DNA, which was accompanied by a marked increase in single-stranded DNA. This DNA damage is dependent on CDK1 and -2 as well as the replication proteins MCM2 and CDT1 but not CDC...

  9. Identification of a BET Family Bromodomain/Casein Kinase II/TAF-Containing Complex as a Regulator of Mitotic Condensin Function

    OpenAIRE

    Hyun-Soo Kim; Rituparna Mukhopadhyay; Scott B. Rothbart; Andrea C. Silva; Vincent Vanoosthuyse; Ernest Radovani; Thomas Kislinger; Assen Roguev; Colm J. Ryan; Jiewei Xu; Harlizawati Jahari; Kevin G. Hardwick; Jack F. Greenblatt; Nevan J. Krogan; Jeffrey S. Fillingham

    2014-01-01

    Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), casein kinase II (CKII), and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions but only briefly colocalize during the periods of chromosome condensation and decondensation. This pattern...

  10. Phosphatidylinositol 3-kinase/Akt pathway regulates hepatic stellate cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Yan Wang; Xiao-Yu Jiang; Li Liu; Hui-Qing Jiang

    2008-01-01

    AIM:To investigate the role of phosphatidylinositol 3-kinase(PI 3-K)/Akt signaling pathway in the balance of HSC activation and apoptosis in rat hepatic stellate cells(HSC).METHODS:An activated HSC cell line was used in this study.LY 294002,the PI 3-K/Akt signal pathway blocker was used to investigate the molecular events on apoptosis in HSC and to interpret the role of this pathway in HSC apoptosis.Immunocytochemistry,Western blot and reverse transcription polymerase chain reaction(RT-PCR)analysis were applied to detect the expression of PI 3-K,and simultaneously phosphorylated-Akt(p-Akt)and total-Akt were determined by Western blot.The HSC apoptosis was examined by annexin-V/propidium iodide double-labelled flow cytometry and transmission electron microscopy.RESULTS:The apoptosis rates in LY 294002(30.82% ±2.90%)and LY 294002+PDGF-BB(28.16%±2.58%)groups were significantly increased compared with those of control(9.02%±1.81%)and PDGF-BB(4.35%±1.18%).PDGF-BB augmented PI 3-K and p-Akt expression.LY 294002 significantly reduced the contents of PI 3-K and p-Akt.mRNA transcription evaluated by RT-PCR showed similar tendencies as protein expression.CONCLUSION:Inhibition of PI 3-K/Akt signaling pathway Induces apoptosis in HSC.(C)2008 The WJG Press.All rights reserved.

  11. Ewing sarcoma EWS protein regulates midzone formation by recruiting Aurora B kinase to the midzone.

    Science.gov (United States)

    Park, Hyewon; Turkalo, Timothy K; Nelson, Kayla; Folmsbee, Stephen Sai; Robb, Caroline; Roper, Brittany; Azuma, Mizuki

    2014-01-01

    Ewing sarcoma is a malignant bone cancer that primarily occurs in children and adolescents. Eighty-five percent of Ewing sarcoma is characterized by the presence of the aberrant chimeric EWS/FLI1 fusion gene. Previously, we demonstrated that an interaction between EWS/FLI1 and wild-type EWS led to the inhibition of EWS activity and mitotic dysfunction. Although defective mitosis is considered to be a critical step in cancer initiation, it is unknown how interference with EWS contributes to Ewing sarcoma formation. Here, we demonstrate that EWS/FLI1- and EWS-knockdown cells display a high incidence of defects in the midzone, a midline structure located between segregating chromatids during anaphase. Defects in the midzone can lead to the failure of cytokinesis and can result in the induction of aneuploidy. The similarity among the phenotypes of EWS/FLI1- and EWS siRNA-transfected HeLa cells points to the inhibition of EWS as the key mechanism for the induction of midzone defects. Supporting this observation, the ectopic expression of EWS rescues the high incidence of midzone defects observed in Ewing sarcoma A673 cells. We discovered that EWS interacts with Aurora B kinase, and that EWS is also required for recruiting Aurora B to the midzone. A domain analysis revealed that the R565 in the RGG3 domain of EWS is essential for both Aurora B interaction and the recruitment of Aurora B to the midzone. Here, we propose that the impairment of EWS-dependent midzone formation via the recruitment of Aurora B is a potential mechanism of Ewing sarcoma development.

  12. Protein kinase C θ regulates the phenotype of murine CD4+ Th17 cells.

    Directory of Open Access Journals (Sweden)

    Katarzyna Wachowicz

    Full Text Available Protein kinase C θ (PKCθ is involved in signaling downstream of the T cell antigen receptor (TCR and is important for shaping effector T cell functions and inflammatory disease development. Acquisition of Th1-like effector features by Th17 cells has been linked to increased pathogenic potential. However, the molecular mechanisms underlying Th17/Th1 phenotypic instability remain largely unknown. In the current study, we address the role of PKCθ in differentiation and function of Th17 cells by using genetic knock-out mice. Implementing in vitro (polarizing T cell cultures and in vivo (experimental autoimmune encephalomyelitis model, EAE techniques, we demonstrated that PKCθ-deficient CD4+ T cells show normal Th17 marker gene expression (interleukin 17A/F, RORγt, accompanied by enhanced production of the Th1-typical markers such as interferon gamma (IFN-γ and transcription factor T-bet. Mechanistically, this phenotype was linked to aberrantly elevated Stat4 mRNA levels in PKCθ-/- CD4+ T cells during the priming phase of Th17 differentiation. In contrast, transcription of the Stat4 gene was suppressed in Th17-primed wild-type cells. This change in cellular effector phenotype was reflected in vivo by prolonged neurological impairment of PKCθ-deficient mice during the course of EAE. Taken together, our data provide genetic evidence that PKCθ is critical for stabilizing Th17 cell phenotype by selective suppression of the STAT4/IFN-γ/T-bet axis at the onset of differentiation.

  13. Oxidized calmodulin kinase II regulates conduction following myocardial infarction: a computational analysis.

    Directory of Open Access Journals (Sweden)

    Matthew D Christensen

    2009-12-01

    Full Text Available Calmodulin kinase II (CaMKII mediates critical signaling pathways responsible for divergent functions in the heart including calcium cycling, hypertrophy and apoptosis. Dysfunction in the CaMKII signaling pathway occurs in heart disease and is associated with increased susceptibility to life-threatening arrhythmia. Furthermore, CaMKII inhibition prevents cardiac arrhythmia and improves heart function following myocardial infarction. Recently, a novel mechanism for oxidative CaMKII activation was discovered in the heart. Here, we provide the first report of CaMKII oxidation state in a well-validated, large-animal model of heart disease. Specifically, we observe increased levels of oxidized CaMKII in the infarct border zone (BZ. These unexpected new data identify an alternative activation pathway for CaMKII in common cardiovascular disease. To study the role of oxidation-dependent CaMKII activation in creating a pro-arrhythmia substrate following myocardial infarction, we developed a new mathematical model of CaMKII activity including both oxidative and autophosphorylation activation pathways. Computer simulations using a multicellular mathematical model of the cardiac fiber demonstrate that enhanced CaMKII activity in the infarct BZ, due primarily to increased oxidation, is associated with reduced conduction velocity, increased effective refractory period, and increased susceptibility to formation of conduction block at the BZ margin, a prerequisite for reentry. Furthermore, our model predicts that CaMKII inhibition improves conduction and reduces refractoriness in the BZ, thereby reducing vulnerability to conduction block and reentry. These results identify a novel oxidation-dependent pathway for CaMKII activation in the infarct BZ that may be an effective therapeutic target for improving conduction and reducing heterogeneity in the infarcted heart.

  14. Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.

    Science.gov (United States)

    Douglas, Pauline; Ye, Ruiqiong; Trinkle-Mulcahy, Laura; Neal, Jessica A; De Wever, Veerle; Morrice, Nick A; Meek, Katheryn; Lees-Miller, Susan P

    2014-01-01

    The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs' role in mitosis may be mechanistically distinct from its well-established role in NHEJ.

  15. Cyclic AMP and AKAP-mediated targeting of protein kinase A regulates lactate dehydrogenase subunit A mRNA stability.

    Science.gov (United States)

    Jungmann, Richard A; Kiryukhina, Olga

    2005-07-01

    Expression of the lactate dehydrogenase A subunit (ldh-A) gene is controlled through transcriptional as well as post-transcriptional mechanisms. Both mechanisms involve activation of protein kinase A (PKA) into its subunits and subsequent phosphorylation and activation of several key regulatory factors. In rat C6 glioma cells, post-transcriptional gene regulation occurs through PKA-mediated stabilization of LDH-A mRNA and subsequent increase of intracellular LDH-A mRNA levels. Previous studies have demonstrated a cAMP-stabilizing region (CSR) located in the LDH-A 3'-untranslated region which, in combination with several phosphorylated CSR-binding proteins (CSR-BP), regulates the PKA-mediated stabilization of LDH-A mRNA. However, the mechanistic details of interaction of CSR with proteins as they pertain to mRNA stabilization by PKA are so far largely unknown. In this study we tested the hypothesis that ribosomal protein extracts (RSW) from glioma cells contain PKA regulatory (RII) and catalytic (C) subunits that, in combination with a protein kinase A anchoring protein (AKAP 95) and CSR-BPs participate in forming CSR-protein complexes that are responsible for mRNA stability regulation. To demonstrate the importance of CSR-protein complex formation, the PKA subunits and AKAP 95 were removed from the RSW by immunoprecipitation, and the antigen-deleted RSW were subjected to CSR binding analysis using gel mobility shift and UV cross-linking. It was shown that AKAP 95 as well as RII formed a direct linkage with CSR during CSR-protein complex formation. In contrast, the catalytic subunit formed part of the CSR-protein complex but did not bind to CSR directly in a covalent linkage. To determine whether formation of CSR complexes that included C, RII, and AKAP 95 constituted a functional event and was necessary for mRNA stabilization, cell-free decay reactions were carried out with RSW extracts, and the kinetics of decay of LDH-A mRNA was determined. Depletion of PKA

  16. Extracellular signal-regulated kinase 1/2 signalling in SLE T cells is influenced by oestrogen and disease activity.

    Science.gov (United States)

    Gorjestani, S; Rider, V; Kimler, B F; Greenwell, C; Abdou, N I

    2008-06-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease that occurs primarily in women of reproductive age. The disease is characterized by exaggerated T-cell activity and abnormal T-cell signalling. The mitogen-activated protein kinase (MAPK) pathway is involved in the maintenance of T-cell tolerance that fails in patients with SLE. Oestrogen is a female sex hormone that binds to nuclear receptors and alters the rate of gene transcription. Oestrogen can also act through the plasma membrane and rapidly stimulate second messengers including calcium flux and kinase activation. In this study, we investigated whether oestrogen influences the activation of MAPK signalling through the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in activated SLE T cells. SLE and control T cells were cultured in serum-free medium without and with oestradiol (10(-7) M) for 18 h. The T cells were activated with phorbol 12 myristate 13-acetate and ionomycin for various time points (0-60 min), and the amount of phosphorylated ERK1/2 was measured by immunoblotting. There were no differences in ERK1/2 phosphorylation between SLE and control T cells at 5 and 15 min after the activation stimulus. However, comparison between the amount of phosphorylated ERK1/2 in SLE T cells from the same patients cultured without and with oestradiol showed a significant oestrogen-dependent suppression (P=0.48) of ERK1/2 in patients with inactive/mild systemic lupus erythematosus disease activity index (SLEDAI) (0-2) compared with patients with moderate (4-6) or active (8-12) SLEDAI scores. These results suggest that the suppression of MAPK through ERK1/2 phosphorylation is sensitive to oestradiol in patients with inactive or mild disease, but the sensitivity is not maintained when disease activity increases. Furthermore, studies are now necessary to understand the mechanisms by which oestrogen influences MAPK activation in SLE T cells. PMID:18539708

  17. Ribosomal protein S6 kinase1 coordinates with TOR-Raptor2 to regulate thylakoid membrane biosynthesis in rice.

    Science.gov (United States)

    Sun, Linxiao; Yu, Yonghua; Hu, Weiqin; Min, Qiming; Kang, Huiling; Li, Yilu; Hong, Yue; Wang, Xuemin; Hong, Yueyun

    2016-07-01

    Ribosomal protein S6 kinase (S6K) functions as a key component in the target of rapamycin (TOR) pathway involved in multiple processes in eukaryotes. The role and regulation of TOR-S6K in lipid metabolism remained unknown in plants. Here we provide genetic and pharmacological evidence that TOR-Raptor2-S6K1 is important for thylakoid galactolipid biosynthesis and thylakoid grana modeling in rice (Oryza sativa L.). Genetic suppression of S6K1 caused pale yellow-green leaves, defective thylakoid grana architecture. S6K1 directly interacts with Raptor2, a core component in TOR signaling, and S6K1 activity is regulated by Raptor2 and TOR. Plants with suppressed Raptor2 expression or reduced TOR activity by inhibitors mimicked the S6K1-deficient phenotype. A significant reduction in galactolipid content was found in the s6k1, raptor2 mutant or TOR-inhibited plants, which was accompanied by decreased transcript levels of the set of genes such as lipid phosphate phosphatase α5 (LPPα5), MGDG synthase 1 (MGD1), and DGDG synthase 1 (DGD1) involved in galactolipid synthesis, compared to the control plants. Moreover, loss of LPPα5 exhibited a similar phenotype with pale yellow-green leaves. These results suggest that TOR-Raptor2-S6K1 is important for modulating thylakoid membrane lipid biosynthesis, homeostasis, thus enhancing thylakoid grana architecture and normal photosynthesis ability in rice. PMID:27102613

  18. Ribosomal protein S6 kinase1 coordinates with TOR-Raptor2 to regulate thylakoid membrane biosynthesis in rice.

    Science.gov (United States)

    Sun, Linxiao; Yu, Yonghua; Hu, Weiqin; Min, Qiming; Kang, Huiling; Li, Yilu; Hong, Yue; Wang, Xuemin; Hong, Yueyun

    2016-07-01

    Ribosomal protein S6 kinase (S6K) functions as a key component in the target of rapamycin (TOR) pathway involved in multiple processes in eukaryotes. The role and regulation of TOR-S6K in lipid metabolism remained unknown in plants. Here we provide genetic and pharmacological evidence that TOR-Raptor2-S6K1 is important for thylakoid galactolipid biosynthesis and thylakoid grana modeling in rice (Oryza sativa L.). Genetic suppression of S6K1 caused pale yellow-green leaves, defective thylakoid grana architecture. S6K1 directly interacts with Raptor2, a core component in TOR signaling, and S6K1 activity is regulated by Raptor2 and TOR. Plants with suppressed Raptor2 expression or reduced TOR activity by inhibitors mimicked the S6K1-deficient phenotype. A significant reduction in galactolipid content was found in the s6k1, raptor2 mutant or TOR-inhibited plants, which was accompanied by decreased transcript levels of the set of genes such as lipid phosphate phosphatase α5 (LPPα5), MGDG synthase 1 (MGD1), and DGDG synthase 1 (DGD1) involved in galactolipid synthesis, compared to the control plants. Moreover, loss of LPPα5 exhibited a similar phenotype with pale yellow-green leaves. These results suggest that TOR-Raptor2-S6K1 is important for modulating thylakoid membrane lipid biosynthesis, homeostasis, thus enhancing thylakoid grana architecture and normal photosynthesis ability in rice.

  19. Matrine-induced autophagy