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Sample records for modulating protein kinase

  1. Modulation of the Chromatin Phosphoproteome by the Haspin Protein Kinase*

    PubMed Central

    Maiolica, Alessio; de Medina-Redondo, Maria; Schoof, Erwin M.; Chaikuad, Apirat; Villa, Fabrizio; Gatti, Marco; Jeganathan, Siva; Lou, Hua Jane; Novy, Karel; Hauri, Simon; Toprak, Umut H.; Herzog, Franz; Meraldi, Patrick; Penengo, Lorenza; Turk, Benjamin E.; Knapp, Stefan; Linding, Rune; Aebersold, Ruedi

    2014-01-01

    Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr3 of the histone H3 (H3), which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date, histone H3 is the only confirmed Haspin substrate. We used a combination of biochemical, pharmacological, and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chromatin-associated proteins and identified a Haspin protein-protein interaction network. We determined the Haspin consensus motif and the co-crystal structure of the kinase with the histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the histone H3 residues Arg2 and Lys4 adjacent to the Haspin phosphorylated threonine into acidic binding pockets. This unique conformation of the kinase-substrate complex explains the reported modulation of Haspin activity by methylation of Lys4 of the histone H3. In addition, the identification of the structural basis of substrate recognition and the amino acid sequence preferences of Haspin aided the identification of novel candidate Haspin substrates. In particular, we validated the phosphorylation of Ser137 of the histone variant macroH2A as a target of Haspin kinase activity. MacroH2A Ser137 resides in a basic stretch of about 40 amino acids that is required to stabilize extranucleosomal DNA, suggesting that phosphorylation of Ser137 might regulate the interactions of macroH2A and DNA. Overall, our data suggest that Haspin activity affects the phosphorylation state of proteins involved in gene expression regulation and splicing. PMID:24732914

  2. Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase Pathway

    DTIC Science & Technology

    2014-10-01

    kinase . This grant proposal will explore the resistance to small molecule AKT protein kinase inhibitors mediated by the... molecule AKT protein kinase inhibitors is potentially mediated by the Pim-1 protein kinase , and that unique Pim protein kinase inhibitors that can in...application is essential for the development of this combined chemotherapeutic strategy. 15. SUBJECT TERMS Small Molecule AKT Inhibitors ,

  3. Functional modulation of AMP-activated protein kinase by cereblon.

    PubMed

    Lee, Kwang Min; Jo, Sooyeon; Kim, Hyunyoung; Lee, Jongwon; Park, Chul-Seung

    2011-03-01

    Mutations in cereblon (CRBN), a substrate binding component of the E3 ubiquitin ligase complex, cause a form of mental retardation in humans. However, the cellular proteins that interact with CRBN remain largely unknown. Here, we report that CRBN directly interacts with the α1 subunit of AMP-activated protein kinase (AMPK α1) and inhibits the activation of AMPK activation. The ectopic expression of CRBN reduces phosphorylation of AMPK α1 and, thus, inhibits the enzyme in a nutrient-independent manner. Moreover, AMPK α1 can be potently activated by suppressing endogenous CRBN using CRBN-specific small hairpin RNAs. Thus, CRBN may act as a negative modulator of the AMPK signaling pathway in vivo.

  4. Protein kinase C modulates ventilatory patterning in the developing rat.

    PubMed

    Bandla, H P; Simakajornboon, N; Graff, G R; Gozal, D

    1999-03-01

    Protein kinase C (PKC) mediates important components of signal transduction pathways underlying neuronal excitability and modulates respiratory timing mechanisms in adult rats. To determine ventilatory effects of systemic PKC inhibition during development, whole-body plethysmographic recordings were conducted in 2-3-d (n = 11), 5-6-d (n = 19), 10-12-d (n = 14), and 20-21-d-old (n = 14) rat pups after treatment with vehicle and Ro 32-0432 (100 mg/kg, intraperitoneally). Ro 32-0432 decreased minute ventilation (V E) by 51.0 +/- 5.5% (mean +/- SEM) in youngest pups (p < 0.01) but only 19.1 +/- 6.8% in 20-21-d-old pups (p < 0.01). V E decreases were always due to frequency reductions with tidal volume (VT) remaining unaffected. Respiratory rate decreases primarily resulted from marked expiratory time (TE) prolongations being more pronounced in 2-3-d-old (115.5 +/- 28.9%) compared with 20-21-d old (36.6 +/- 10.9%; p < 0.002 analysis of variance [ANOVA] ). Expression of the PKC isoforms alpha, beta, gamma, delta, iota, and mu was further examined in brainstem and cortex by immunoblotting and revealed different patterns with postnatal age and location. We conclude that endogenous PKC inhibition elicits age-dependent ventilatory reductions which primarily affect timing mechanisms rather than changes in volume drive. This effect on ventilation abates with increasing postnatal age suggesting that the neural substrate mediating overall respiratory output may be more critically dependent on PKC activity in the immature animal.

  5. Rassf Proteins as Modulators of Mst1 Kinase Activity

    PubMed Central

    Bitra, Aruna; Sistla, Srinivas; Mariam, Jessy; Malvi, Harshada; Anand, Ruchi

    2017-01-01

    Rassf1A/5 tumor suppressors serve as adaptor proteins possessing a modular architecture with the C-terminal consisting of a coiled-coil SARAH (Salvador-Rassf-Hippo) domain and the central portion being composed of Ras associated (RA) domain. Here, we investigate the effect of Rassf effectors on Mst1 function by mapping the interaction of various domains of Rassf1A/5 and Mst1 kinase using surface plasmon resonance (SPR). The results revealed that apart from the C-terminal SARAH domain of Mst1 which interacts to form heterodimers with Rassf1A/5, the N-terminal kinase domain of Mst1 plays a crucial role in the stabilization of this complex. In addition, SPR experiments show that the RA domains play an important role in fine-tuning the Mst1-Rassf interaction, with Rassf5 being a preferred partner over a similar Rassf1A construct. It was also demonstrated that the activity profile of Mst1 in presence of Rassf adaptors completely switches. A Rassf-Mst1 complexed version of the kinase becomes apoptotic by positively regulating Mst1-H2B mediated serine 14 histone H2B phosphorylation, a hallmark of chromatin condensation. In contrast, the heterodimerization of Mst1 with Rassf1A/5 suppresses the phosphorylation of FoxO, thereby inhibiting the downstream Mst1-FoxO signalling pathway. PMID:28327630

  6. Small molecule modulators of eukaryotic initiation factor 2α kinases, the key regulators of protein synthesis.

    PubMed

    Joshi, Manali; Kulkarni, Abhijeet; Pal, Jayanta K

    2013-11-01

    Eukaryotic initiation factor 2 alpha kinases (eIF-2α kinases) are key mediators of stress response in cells. In mammalian cells, there are four eIF-2α kinases, namely HRI (Heme-Regulated Inhibitor), PKR (RNA-dependent Protein Kinase), PERK (PKR-like ER Kinase) and GCN2 (General Control Non-derepressible 2). These kinases get activated during diverse cytoplasmic stress conditions and phosphorylate the alpha-subunit of eIF2, leading to global protein synthesis inhibition. Therefore, eIF-2α kinases play a vital role in various cellular processes such as proliferation, differentiation, apoptosis and cell signaling. Deregulation of eIF-2α kinases and protein synthesis has been linked to numerous pathological conditions such as certain cancers, anemia and neurodegenerative disorders. Thus, modulation of these kinases by small molecules holds a great therapeutic promise. In this review we have compiled the available information on inhibitors and activators of these four eIF-2α kinases. The review concludes with a note on the selectivity issue of currently available modulators and future perspectives for the design of specific small molecule probes.

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

    PubMed

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

    2009-02-15

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

  8. Protein kinase A phosphorylation modulates transport of the polypyrimidine tract-binding protein

    PubMed Central

    Xie, Jiuyong; Lee, Ji-Ann; Kress, Tracy L.; Mowry, Kimberly L.; Black, Douglas L.

    2003-01-01

    The heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins play important roles in mRNA processing in eukaryotes, but little is known about how they are regulated by cellular signaling pathways. The polypyrimidine-tract binding protein (PTB, or hnRNP I) is an important regulator of alternative pre-mRNA splicing, of viral RNA translation, and of mRNA localization. Here we show that the nucleo-cytoplasmic transport of PTB is regulated by the 3′,5′-cAMP-dependent protein kinase (PKA). PKA directly phosphorylates PTB on conserved Ser-16, and PKA activation in PC12 cells induces Ser-16 phosphorylation. PTB carrying a Ser-16 to alanine mutation accumulates normally in the nucleus. However, export of this mutant protein from the nucleus is greatly reduced in heterokaryon shuttling assays. Conversely, hyperphosphorylation of PTB by coexpression with the catalytic subunit of PKA results in the accumulation of PTB in the cytoplasm. This accumulation is again specifically blocked by the S16A mutation. Similarly, in Xenopus oocytes, the phospho-Ser-16-PTB is restricted to the cytoplasm, whereas the non-Ser-16-phosphorylated PTB is nuclear. Thus, direct PKA phosphorylation of PTB at Ser-16 modulates the nucleo-cytoplasmic distribution of PTB. This phosphorylation likely plays a role in the cytoplasmic function of PTB. PMID:12851456

  9. Glucose regulates diacylglycerol intracellular levels and protein kinase C activity by modulating diacylglycerol kinase subcellular localization.

    PubMed

    Miele, Claudia; Paturzo, Flora; Teperino, Raffaele; Sakane, Fumio; Fiory, Francesca; Oriente, Francesco; Ungaro, Paola; Valentino, Rossella; Beguinot, Francesco; Formisano, Pietro

    2007-11-02

    Although chronic hyperglycemia reduces insulin sensitivity and leads to impaired glucose utilization, short term exposure to high glucose causes cellular responses positively regulating its own metabolism. We show that exposure of L6 myotubes overexpressing human insulin receptors to 25 mm glucose for 5 min decreased the intracellular levels of diacylglycerol (DAG). This was paralleled by transient activation of diacylglycerol kinase (DGK) and of insulin receptor signaling. Following 30-min exposure, however, both DAG levels and DGK activity returned close to basal levels. Moreover, the acute effect of glucose on DAG removal was inhibited by >85% by the DGK inhibitor R59949. DGK inhibition was also accompanied by increased protein kinase C-alpha (PKCalpha) activity, reduced glucose-induced insulin receptor activation, and GLUT4 translocation. Glucose exposure transiently redistributed DGK isoforms alpha and delta, from the prevalent cytosolic localization to the plasma membrane fraction. However, antisense silencing of DGKdelta, but not of DGKalpha expression, was sufficient to prevent the effect of high glucose on PKCalpha activity, insulin receptor signaling, and glucose uptake. Thus, the short term exposure of skeletal muscle cells to glucose causes a rapid induction of DGK, followed by a reduction of PKCalpha activity and transactivation of the insulin receptor signaling. The latter may mediate, at least in part, glucose induction of its own metabolism.

  10. Small molecule adenosine 5'-monophosphate activated protein kinase (AMPK) modulators and human diseases.

    PubMed

    Rana, Sandeep; Blowers, Elizabeth C; Natarajan, Amarnath

    2015-01-08

    Adenosine 5'-monophosphate activated protein kinase (AMPK) is a master sensor of cellular energy status that plays a key role in the regulation of whole-body energy homeostasis. AMPK is a serine/threonine kinase that is activated by upstream kinases LKB1, CaMKKβ, and Tak1, among others. AMPK exists as αβγ trimeric complexes that are allosterically regulated by AMP, ADP, and ATP. Dysregulation of AMPK has been implicated in a number of metabolic diseases including type 2 diabetes mellitus and obesity. Recent studies have associated roles of AMPK with the development of cancer and neurological disorders, making it a potential therapeutic target to treat human diseases. This review focuses on the structure and function of AMPK, its role in human diseases, and its direct substrates and provides a brief synopsis of key AMPK modulators and their relevance in human diseases.

  11. Virtual screening of protein kinase C inhibitors from natural product library to modulate general anaesthetic effects.

    PubMed

    Zhao, Junhui; Zhou, Chuixian

    2015-01-01

    Protein kinase C (PKC) plays a key role in neurotransmission in the central nervous system, and targeting PKC domain is considered as a strategy to modulate the anaesthetic effects. In this study, we described a synthetic pipeline to perform high-throughput virtual screening against a large library of 3D structural natural products released recently in order to discover those potential PKC modulators. A total of 100 natural products with top scores were raised, from which 12 promising candidates were tested to determine their inhibitory potencies against PKC. As might be expected, the promiscuous kinase inhibitor staurosporine showed a high PKC inhibitory activity (IC50 = 64 nM), and other two tested compounds, i.e. fisetin and tetrahydropapaverine, were also highly potent with their activities at nanomolar level (IC50 = 370 and 190, respectively).

  12. A role for cyclin-dependent kinase(s) in the modulation of fast anterograde axonal transport: effects defined by olomoucine and the APC tumor suppressor protein

    NASA Technical Reports Server (NTRS)

    Ratner, N.; Bloom, G. S.; Brady, S. T.

    1998-01-01

    Proteins that interact with both cytoskeletal and membrane components are candidates to modulate membrane trafficking. The tumor suppressor proteins neurofibromin (NF1) and adenomatous polyposis coli (APC) both bind to microtubules and interact with membrane-associated proteins. The effects of recombinant NF1 and APC fragments on vesicle motility were evaluated by measuring fast axonal transport along microtubules in axoplasm from squid giant axons. APC4 (amino acids 1034-2844) reduced only anterograde movements, whereas APC2 (aa 1034-2130) or APC3 (aa 2130-2844) reduced both anterograde and retrograde transport. NF1 had no effect on organelle movement in either direction. Because APC contains multiple cyclin-dependent kinase (CDK) consensus phosphorylation motifs, the kinase inhibitor olomoucine was examined. At concentrations in which olomoucine is specific for cyclin-dependent kinases (5 microM), it reduced only anterograde transport, whereas anterograde and retrograde movement were both affected at concentrations at which other kinases are inhibited as well (50 microM). Both anterograde and retrograde transport also were inhibited by histone H1 and KSPXK peptides, substrates for proline-directed kinases, including CDKs. Our data suggest that CDK-like axonal kinases modulate fast anterograde transport and that other axonal kinases may be involved in modulating retrograde transport. The specific effect of APC4 on anterograde transport suggests a model in which the binding of APC to microtubules may limit the activity of axonal CDK kinase or kinases in restricted domains, thereby affecting organelle transport.

  13. Extract of Reishi polysaccharides induces cytokine expression via TLR4-modulated protein kinase signaling pathways.

    PubMed

    Hsu, Hsien-Yeh; Hua, Kuo-Feng; Lin, Chun-Cheng; Lin, Chun-Hung; Hsu, Jason; Wong, Chi-Huey

    2004-11-15

    We have demonstrated that an extract of Ganoderma lucidum (Reishi or Ling-Zhi) polysaccharides (EORP) exerts immunomodulating activities by stimulating the expression of inflammatory cytokines from mouse spleen cells. Interestingly, via responding to LPS in genetic variation of murine macrophage HeNC2 and GG2EE cell lines, and using TLR4 Ab blockage in human blood-derived monocytic macrophages, we have found that the TLR4, but not complement receptor type 3, is a putative receptor of EORP, mediating the consequent immunomodulating events associated with IL-1 gene expression. Based on our studies of reactive oxygen species production, polymyxin B inhibition, and protein tyrosine kinase (PTK) activity, we ruled out the possibility of LPS contamination in EORP. We have found that EORP differentially modulates the protein kinase (PK)-mediated signal transduction pathways associated with inflammatory cytokine IL-1. In human macrophages and murine macrophage J774A.1 cells, EORP was found to up-regulate IL-1 secretion and pro-IL-1 (precursor of IL-1) as well as IL-1-converting enzyme expression. Specifically, EORP rapidly stimulates PTK-mediated phosphorylation, followed by induction of PKs and activation of MAPKs: ERK, JNK, and p38. Using PK inhibitors in the kinase activity assays, Western blot analyses and IL-1 ELISA, we have extensively examined and dissected the role of individual PK in the regulation of pro-IL-1/IL-1. Our findings establish that EORP-mediated signaling pathways are involved in the pro-IL-1/IL-1 regulation: PTK/protein kinase C/MEK1/ERK and PTK/Rac1/p21-activated kinase/p38.

  14. Isozyme-Specific Effects of Protein Kinase C in Pain Modulation

    PubMed Central

    Zhao, Chengshui; Leitges, Michael; Gereau, Robert W.

    2011-01-01

    Background Protein kinase C (PKC) is a family of serine/threonine kinases that contains more than 10 isozymes. Evidence suggests that PKC may play important roles in pain modulation, but the isozyme-specific effects of PKC on different aspects of pain modulation are not fully understood. We hypothesize that different PKC isozymes play different roles in different aspects of pain modulation. Methods The nociceptive behaviors of mice with deletion of PKC α, β, γ, or δ in multiple pain models were compared with their respective wild type littermates. Also, the morphine analgesia and the development of morphine tolerance in mice with deletion of PKC γ were compared with their respective wild type littermates. Results Thermal hyperalgesia induced by complete Freund’s adjuvant injection was significantly attenuated by the deletion of PKC β, γ or δ, but not PKC α. Deletion of PKC γ significantly attenuated neuropathic mechanical allodynia induced by spared nerve injury, whereas deletion of PKC α enhanced this allodynia. Baseline thermal and mechanical sensitivity, nociceptive behaviors induced by formalin, mechanical allodynia induced by complete Freund’s adjuvant injection, were not altered by deletion of PKC α, β, γ or δ. Finally, morphine analgesia and the development of morphine tolerance were not altered in PKC γ-deficient mice. Conclusions PKC plays isozyme-specific effects in pain modulation. PMID:22042410

  15. Overcoming Resistance to Inhibitors of the AKT Protein Kinase by Modulation of the Pim Kinase Pathway

    DTIC Science & Technology

    2013-10-01

    translation of the MET receptor tyrosine kinase in prostate cancer. This regulates the activity of the MET/ HGF axis and potentially can affect the...on culture of wild-type DU145 cells in the presence of HGF was enhanced in the Pim-1-overexpressing cells (Fig 6a). This effect was specific as there...was no difference in ERK phosphorylation between the over expressor and wild-type cell lines cultured in HGF . Conversely, in PC3-LN4 cells in

  16. Lithium blocks ethanol-induced modulation of protein kinases in the developing brain

    SciTech Connect

    Chakraborty, Goutam; Saito, Mitsuo; Mao, Rui-Fen; Wang, Ray; Vadasz, Csaba; Saito, Mariko

    2008-03-14

    Lithium has been shown to be neuroprotective against various insults including ethanol exposure. We previously reported that ethanol-induced apoptotic neurodegeneration in the postnatal day 7 (P7) mice is associated with decreases in phosphorylation levels of Akt, glycogen synthase kinase-3{beta} (GSK-3{beta}), and AMP-activated protein kinase (AMPK), and alteration in lipid profiles in the brain. Here, P7 mice were injected with ethanol and lithium, and the effects of lithium on ethanol-induced alterations in phosphorylation levels of protein kinases and lipid profiles in the brain were examined. Immunoblot and immunohistochemical analyses showed that lithium significantly blocked ethanol-induced caspase-3 activation and reduction in phosphorylation levels of Akt, GSK-3{beta}, and AMPK. Further, lithium inhibited accumulation of cholesterol ester (ChE) and N-acylphosphatidylethanolamine (NAPE) triggered by ethanol in the brain. These results suggest that Akt, GSK-3{beta}, and AMPK are involved in ethanol-induced neurodegeneration and the neuroprotective effects of lithium by modulating both apoptotic and survival pathways.

  17. Reishi immuno-modulation protein induces interleukin-2 expression via protein kinase-dependent signaling pathways within human T cells.

    PubMed

    Hsu, Hsien-Yeh; Hua, Kuo-Feng; Wu, Wei-Chi; Hsu, Jason; Weng, Shih-Ting; Lin, Tsai-Leng; Liu, Chun-Yi; Hseu, Ruey-Shyang; Huang, Ching-Tsan

    2008-04-01

    Ganoderma lucidum, a medicinal fungus is thought to possess and enhance a variety of human immune functions. An immuno-modulatory protein, Ling Zhi-8 (LZ-8) isolated from G. lucidum exhibited potent mitogenic effects upon human peripheral blood lymphocytes (PBL). However, LZ-8-mediated signal transduction in the regulation of interleukin-2 (IL-2) gene expression within human T cells is largely unknown. Here we cloned the LZ-8 gene of G. lucidum, and expressed the recombinant LZ-8 protein (rLZ-8) by means of a yeast Pichia pastoris protein expression system. We found that rLZ-8 induces IL-2 gene expression via the Src-family protein tyrosine kinase (PTK), via reactive oxygen species (ROS), and differential protein kinase-dependent pathways within human primary T cells and cultured Jurkat T cells. In essence, we have established the nature of the rLZ-8-mediated signal-transduction pathways, such as PTK/protein kinase C (PKC)/ROS, PTK/PLC/PKCalpha/ERK1/2, and PTK/PLC/PKCalpha/p38 pathways in the regulation of IL-2 gene expression within human T cells. Our current results of analyzing rLZ-8-mediated signal transduction in T cells might provide a potential application for rLZ-8 as a pharmacological immune-modulating agent.

  18. Modulation of Leishmania major aquaglyceroporin activity by a mitogen-activated protein kinase

    PubMed Central

    Mandal, Goutam; Sharma, Mansi; Kruse, Martin; Sander-Juelch, Claudia; Munro, Laura Anne; Wang, Yong; Vilg, Jenny Veide; Tamás, Markus J; Bhattacharjee, Hiranmoy; Wiese, Martin; Mukhopadhyay, Rita

    2012-01-01

    Summary Leishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defense against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogen-activated protein kinase, LmjMPK2. Leishmania parasites co-expressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo-osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr197 and this phosphorylation requires LmjMPK2 activity. Lys42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. L. mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild type cells. This is the first report where a parasite aquaglyceroporin activity is post-translationally modulated by a MAP kinase. PMID:22779703

  19. Myofibrillar Z-discs Are a Protein Phosphorylation Hot Spot with Protein Kinase C (PKCα) Modulating Protein Dynamics.

    PubMed

    Reimann, Lena; Wiese, Heike; Leber, Yvonne; Schwäble, Anja N; Fricke, Anna L; Rohland, Anne; Knapp, Bettina; Peikert, Christian D; Drepper, Friedel; van der Ven, Peter F M; Radziwill, Gerald; Fürst, Dieter O; Warscheid, Bettina

    2017-03-01

    The Z-disc is a protein-rich structure critically important for the development and integrity of myofibrils, which are the contractile organelles of cross-striated muscle cells. We here used mouse C2C12 myoblast, which were differentiated into myotubes, followed by electrical pulse stimulation (EPS) to generate contracting myotubes comprising mature Z-discs. Using a quantitative proteomics approach, we found significant changes in the relative abundance of 387 proteins in myoblasts versus differentiated myotubes, reflecting the drastic phenotypic conversion of these cells during myogenesis. Interestingly, EPS of differentiated myotubes to induce Z-disc assembly and maturation resulted in increased levels of proteins involved in ATP synthesis, presumably to fulfill the higher energy demand of contracting myotubes. Because an important role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape further warranted in-depth analysis. We therefore established, by global phosphoproteomics of EPS-treated contracting myotubes, a comprehensive site-resolved protein phosphorylation map of the Z-disc and found that it is a phosphorylation hotspot in skeletal myocytes, underscoring its functions in signaling and disease-related processes. In an illustrative fashion, we analyzed the actin-binding multiadaptor protein filamin C (FLNc), which is essential for Z-disc assembly and maintenance, and found that PKCα phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. Fluorescence recovery after photobleaching experiments indicated that this phosphorylation modulates FLNc dynamics. Moreover, FLNc lacking the cleaved Ig-like domain 24 exhibited remarkably fast kinetics and exceedingly high mobility. Our data set provides research community resource for further identification of kinase-mediated changes in myofibrillar protein interactions

  20. G-Protein-Coupled Receptor Kinase 2 as a Potential Modulator of the Hallmarks of Cancer.

    PubMed

    Nogués, Laura; Reglero, Clara; Rivas, Verónica; Neves, María; Penela, Petronila; Mayor, Federico

    2017-03-01

    Malignant features-such as sustained proliferation, refractoriness to growth suppressors, resistance to cell death or aberrant motility, and metastasis-can be triggered by a variety of mutations and signaling adaptations. Signaling nodes can act as cancer-associated factors by cooperating with oncogene-governed pathways or participating in compensatory transduction networks to strengthen tumor properties. G-protein-coupled receptor kinase 2 (GRK2) is arising as one of such nodes. Via its complex network of connections with other cellular proteins, GRK2 contributes to the modulation of basic cellular functions-such as cell proliferation, survival, or motility-and is involved in metabolic homeostasis, inflammation, or angiogenic processes. Moreover, altered GRK2 levels are starting to be reported in different tumoral contexts and shown to promote breast tumorigenesis or to trigger the tumoral angiogenic switch. The ability to modulate several of the hallmarks of cancer puts forward GRK2 as an oncomodifier, able to modulate carcinogenesis in a cell-type specific way.

  1. Pharmacological modulation of protein kinases as a new approach to treat addiction to cocaine and opiates.

    PubMed

    García-Pardo, María Pilar; Roger-Sanchez, Concepción; Rodríguez-Arias, Marta; Miñarro, Jose; Aguilar, María Asunción

    2016-06-15

    Drug addiction shares brain mechanisms and molecular substrates with learning and memory processes, such as the stimulation of glutamate receptors and their downstream signalling pathways. In the present work we provide an up-to-date review of studies that have demonstrated the implication of the main memory-related calcium-dependent protein kinases in opiate and cocaine addiction. The effects of these drugs of abuse in different animal models of drug reward, dependence and addiction are altered by manipulation of the mitogen-activated protein kinase (MAPK) family, particularly extracellular signal regulated kinase (ERK), calcium/calmodulin-dependent kinase II (CaMKII), the protein kinase C (PKC) family (including PKMζ), cAMP-dependent protein kinase A (PKA), cGMP-dependent protein kinase G (PKG), the phosphatidylinositol 3-kinase (PI3K) pathway and its downstream target mammalian target of Rapamycin (mTOR), cyclin-dependent kinase 5 (Cdk5), heat-shock proteins (Hsp) and other enzymes and proteins. Research suggests that drugs of abuse induce dependence and addiction by modifying the signalling pathways that involve these memory-related protein kinases, and supports the idea that drug addiction is an excessive aberrant learning disorder in which the maladaptive memory of drug-associated cues maintains compulsive drug use and contributes to relapse. Moreover, the studies we review offer new pharmacological strategies to treat opiate and cocaine dependence based on the manipulation of these protein kinases. In particular, disruption of reconsolidation of drug-related memories may have a high therapeutic value in the treatment of drug addiction.

  2. Modulation of PDT-induced apoptosis by protein kinases and phosphatases

    NASA Astrophysics Data System (ADS)

    Luo, Yu; Chang, Chi K.; Kessel, David

    1996-04-01

    Photodynamic therapy of neoplastic cell lines can lead to the rapid initiation of apoptosis, a mode of cell death that results in a characteristic pattern of cellular and DNA fragmentation. In this study, we examine the effects of protein tyrosine- and serine/threonine phosphatases and kinases on the fragmentation of DNA to 50 kb and photodynamic effects of lysosomal and mitochondrial photosensitizers on murine leukemia P388 cells. The data are consistent with the proposal that maintenance of phosphorylated tyrosine residues is essential for the PDT- induced processing of 50 kb DNA to nucleosomes, while maintenance of serine phosphorylation inhibits such processing. Factors involved in chromatin fragmentation to 50 kb particles have yet to be elucidated. Several agents which mediate membrane photodamage mimic the effect of protein serine/threonine phosphatase inhibitors, i.e., they inhibit further processing of the 50 kb DNA formed as a consequence of lysosomal or mitochondrial photodamage. These results indicate that even the rapid initiation of apoptosis by PDT is modulated by phosphatase and kinase activities.

  3. Modulation of human c-mpl gene expression by thrombopoietin through protein kinase C.

    PubMed

    Sunohara, M; Morikawa, S; Sato, T; Sato, I; Sato, T; Fuse, A

    2003-01-01

    The c-Mpl, thrombopoietin (TPO) receptor specificially controls megakaryocytic growth and differentiation. TPO increased the c-mpl promoter activity determined by a transient expression system using a vector containing the luciferase gene as a reporter in the human megakaryoblastic cell line CMK. The maximal promoter activity of c-mpl was obtained 24 hr after pretreatment with TPO for 3 hr and then declined with time. This increase was completely abolished by protein kinase C (PKC) inhibitors (GF109203, calphostin C and H7). Phorbol 12-myristate 13-acetate (PMA) treatment led to an increase in c-mpl promoter activity. These results demonstrate that the promoter activity of c-mpl is modulated by transcription through a PKC-dependent pathway.

  4. Complexes between STE5 and components of the pheromone-responsive mitogen-activated protein kinase module.

    PubMed Central

    Marcus, S; Polverino, A; Barr, M; Wigler, M

    1994-01-01

    We present genetic evidence for complex formation of STE5 and the STE11, STE7, and FUS3 protein kinases, the pheromone-responsive mitogen-activated protein kinase module of Saccharomyces cerevisiae. Interaction between STE5 and STE11 is not dependent on STE7, and interaction between STE5 and STE7 does not require STE11. The N-terminal regulatory domain of STE11 is both necessary and sufficient for interaction with STE5. Interaction between STE7 and STE11 is bridged by STE5, suggesting the formation of a multiprotein complex. We also demonstrate biochemical interaction between STE5 and STE11 by using a combination of bacterially expressed fusion proteins and extracts prepared from yeast. Our results suggest that STE5 is a scaffolding protein that facilitates interactions between components of the pheromone-responsive mitogen-activated protein kinase module. We further propose that such scaffolding proteins serve to inhibit cross-talk between functionally unrelated mitogen-activated protein kinase modules within the same cell. Images PMID:8052657

  5. LIG Family Receptor Tyrosine Kinase-Associated Proteins Modulate Growth Factor Signals During Neural Development

    PubMed Central

    Mandai, Kenji; Guo, Ting; Hillaire, Coryse St.; Meabon, James S.; Kanning, Kevin C.; Bothwell, Mark; Ginty, David D.

    2009-01-01

    SUMMARY Genome-wide screens were performed to identify transmembrane proteins that mediate axonal growth, guidance and target field innervation of somatosensory neurons. One gene, Linx (alias Islr2), encoding a leucine-rich repeat and immunoglobulin (LIG) family protein, is expressed in a subset of developing sensory and motor neurons. Domain and genomic structures of Linx and other LIG family members suggest that they are evolutionarily related to Trk receptor tyrosine kinases (RTKs). Several LIGs, including Linx are expressed in subsets of somatosensory and motor neurons and select members interact with TrkA and Ret RTKs. Moreover, axonal projection defects in mice harboring a null mutation in Linx resemble those in mice lacking Ngf, TrkA and Ret. In addition, Linx modulates NGF–TrkA- and GDNF–GFRα1/Ret-mediated axonal extension in cultured sensory and motor neurons, respectively. These findings show that LIGs physically interact with RTKs and modulate their activities to control axonal extension, guidance and branching. PMID:19755105

  6. LIG family receptor tyrosine kinase-associated proteins modulate growth factor signals during neural development.

    PubMed

    Mandai, Kenji; Guo, Ting; St Hillaire, Coryse; Meabon, James S; Kanning, Kevin C; Bothwell, Mark; Ginty, David D

    2009-09-10

    Genome-wide screens were performed to identify transmembrane proteins that mediate axonal growth, guidance and target field innervation of somatosensory neurons. One gene, Linx (alias Islr2), encoding a leucine-rich repeat and immunoglobulin (LIG) family protein, is expressed in a subset of developing sensory and motor neurons. Domain and genomic structures of Linx and other LIG family members suggest that they are evolutionarily related to Trk receptor tyrosine kinases (RTKs). Several LIGs, including Linx, are expressed in subsets of somatosensory and motor neurons, and select members interact with TrkA and Ret RTKs. Moreover, axonal projection defects in mice harboring a null mutation in Linx resemble those in mice lacking Ngf, TrkA, and Ret. In addition, Linx modulates NGF-TrkA- and GDNF-GFRalpha1/Ret-mediated axonal extension in cultured sensory and motor neurons, respectively. These findings show that LIGs physically interact with RTKs and modulate their activities to control axonal extension, guidance and branching.

  7. Coronin 1 Regulates Cognition and Behavior through Modulation of cAMP/Protein Kinase A Signaling

    PubMed Central

    Zhang, Chun-Lei; Moshous, Despina; Studer, Vera; Schneider, Jacques; Genoud, Christel; Fossoud, Catherine; Gambino, Frédéric; Khelfaoui, Malik; Müller, Christian; Bartholdi, Deborah; Rossez, Helene; Stiess, Michael; Houbaert, Xander; Jaussi, Rolf; Frey, Daniel; Kammerer, Richard A.; Deupi, Xavier; de Villartay, Jean-Pierre; Lüthi, Andreas; Humeau, Yann; Pieters, Jean

    2014-01-01

    Cognitive and behavioral disorders are thought to be a result of neuronal dysfunction, but the underlying molecular defects remain largely unknown. An important signaling pathway involved in the regulation of neuronal function is the cyclic AMP/Protein kinase A pathway. We here show an essential role for coronin 1, which is encoded in a genomic region associated with neurobehavioral dysfunction, in the modulation of cyclic AMP/PKA signaling. We found that coronin 1 is specifically expressed in excitatory but not inhibitory neurons and that coronin 1 deficiency results in loss of excitatory synapses and severe neurobehavioral disabilities, including reduced anxiety, social deficits, increased aggression, and learning defects. Electrophysiological analysis of excitatory synaptic transmission in amygdala revealed that coronin 1 was essential for cyclic–AMP–protein kinase A–dependent presynaptic plasticity. We further show that upon cell surface stimulation, coronin 1 interacted with the G protein subtype Gαs to stimulate the cAMP/PKA pathway. The absence of coronin 1 or expression of coronin 1 mutants unable to interact with Gαs resulted in a marked reduction in cAMP signaling. Strikingly, synaptic plasticity and behavioral defects of coronin 1–deficient mice were restored by in vivo infusion of a membrane-permeable cAMP analogue. Together these results identify coronin 1 as being important for cognition and behavior through its activity in promoting cAMP/PKA-dependent synaptic plasticity and may open novel avenues for the dissection of signal transduction pathways involved in neurobehavioral processes. PMID:24667537

  8. Protein kinase C modulates inactivation of Kv3.3 channels.

    PubMed

    Desai, Rooma; Kronengold, Jack; Mei, Jianfeng; Forman, Stuart A; Kaczmarek, Leonard K

    2008-08-08

    Modulation of some Kv3 family potassium channels by protein kinase C (PKC) regulates their amplitude and kinetics and adjusts firing patterns of auditory neurons in response to stimulation. Nevertheless, little is known about the modulation of Kv3.3, a channel that is widely expressed throughout the nervous system and is the dominant Kv3 family member in auditory brainstem. We have cloned the cDNA for the Kv3.3 channel from mouse brain and have expressed it in a mammalian cell line and in Xenopus oocytes to characterize its biophysical properties and modulation by PKC. Kv3.3 currents activate at positive voltages and undergo inactivation with time constants of 150-250 ms. Activators of PKC increased current amplitude and removed inactivation of Kv3.3 currents, and a specific PKC pseudosubstrate inhibitor peptide prevented the effects of the activators. Elimination of the first 78 amino acids of the N terminus of Kv3.3 produced noninactivating currents suggesting that PKC modulates N-type inactivation, potentially by phosphorylation of sites in this region. To identify potential phosphorylation sites, we investigated the response of channels in which serines in this N-terminal domain were subjected to mutagenesis. Our results suggest that serines at positions 3 and 9 are potential PKC phosphorylation sites. Computer simulations of model neurons suggest that phosphorylation of Kv3.3 by PKC may allow neurons to maintain action potential height during stimulation at high frequencies, and may therefore contribute to stimulus-induced changes in the intrinsic excitability of neurons such as those of the auditory brainstem.

  9. Melatonin decreases breast cancer metastasis by modulating Rho-associated kinase protein-1 expression.

    PubMed

    Borin, Thaiz Ferraz; Arbab, Ali Syed; Gelaleti, Gabriela Bottaro; Ferreira, Lívia Carvalho; Moschetta, Marina Gobbe; Jardim-Perassi, Bruna Victorasso; Iskander, A S M; Varma, Nadimpalli Ravi S; Shankar, Adarsh; Coimbra, Verena Benedick; Fabri, Vanessa Alves; de Oliveira, Juliana Garcia; Zuccari, Debora Aparecida Pires de Campos

    2016-01-01

    The occurrence of metastasis, an important breast cancer prognostic factor, depends on cell migration/invasion mechanisms, which can be controlled by regulatory and effector molecules such as Rho-associated kinase protein (ROCK-1). Increased expression of this protein promotes tumor growth and metastasis, which can be restricted by ROCK-1 inhibitors. Melatonin has shown oncostatic, antimetastatic, and anti-angiogenic effects and can modulate ROCK-1 expression. Metastatic and nonmetastatic breast cancer cell lines were treated with melatonin as well as with specific ROCK-1 inhibitor (Y27632). Cell viability, cell migration/invasion, and ROCK-1 gene expression and protein expression were determined in vitro. In vivo lung metastasis study was performed using female athymic nude mice treated with either melatonin or Y27832 for 2 and 5 wk. The metastases were evaluated by X-ray computed tomography and single photon emission computed tomography (SPECT) and by immunohistochemistry for ROCK-1 and cytokeratin proteins. Melatonin and Y27632 treatments reduced cell viability and invasion/migration of both cell lines and decreased ROCK-1 gene expression in metastatic cells and protein expression in nonmetastatic cell line. The numbers of 'hot' spots (lung metastasis) identified by SPECT images were significantly lower in treated groups. ROCK-1 protein expression also was decreased in metastatic foci of treated groups. Melatonin has shown to be effective in controlling metastatic breast cancer in vitro and in vivo, not only via inhibition of the proliferation of tumor cells but also through direct antagonism of metastatic mechanism of cells rendered by ROCK-1 inhibition. When Y27632 was used, the effects were similar to those found with melatonin treatment.

  10. Melatonin decreases breast cancer metastasis by modulating Rho-associated kinase protein-1 expression

    PubMed Central

    Borin, Thaiz Ferraz; Arbab, Ali Syed; Gelaleti, Gabriela Bottaro; Ferreira, Lívia Carvalho; Moschetta, Marina Gobbe; Jardim-Perassi, Bruna Victorasso; Iskander, ASM; Varma, Nadimpalli Ravi S.; Shankar, Adarsh; Coimbra, Verena Benedick; Fabri, Vanessa Alves; de Oliveira, Juliana Garcia; de Campos Zuccari, Debora Aparecida Pires

    2016-01-01

    The occurrence of metastasis, an important breast cancer prognostic factor, depends on cell migration/invasion mechanisms, which can be controlled by regulatory and effector molecules such as Rho-associated kinase protein (ROCK-1). Increased expression of this protein promotes tumor growth and metastasis, which can be restricted by ROCK-1 inhibitors. Melatonin has shown oncostatic, antimetastatic, and anti-angiogenic effects and can modulate ROCK-1 expression. Metastatic and nonmetastatic breast cancer cell lines were treated with melatonin as well as with specific ROCK-1 inhibitor (Y27632). Cell viability, cell migration/invasion, and ROCK-1 gene expression and protein expression were determined in vitro. In vivo lung metastasis study was performed using female athymic nude mice treated with either melatonin or Y27832 for 2 and 5 wk. The metastases were evaluated by X-ray computed tomography and single photon emission computed tomography (SPECT) and by immunohistochemistry for ROCK-1 and cytokeratin proteins. Melatonin and Y27632 treatments reduced cell viability and invasion/migration of both cell lines and decreased ROCK-1 gene expression in metastatic cells and protein expression in nonmetastatic cell line. The numbers of ‘hot’ spots (lung metastasis) identified by SPECT images were significantly lower in treated groups. ROCK-1 protein expression also was decreased in metastatic foci of treated groups. Melatonin has shown to be effective in controlling metastatic breast cancer in vitro and in vivo, not only via inhibition of the proliferation of tumor cells but also through direct antagonism of metastatic mechanism of cells rendered by ROCK-1 inhibition. When Y27632 was used, the effects were similar to those found with melatonin treatment. PMID:26292662

  11. Protein kinase A modulation of CaV1.4 calcium channels

    PubMed Central

    Sang, Lingjie; Dick, Ivy E.; Yue, David T.

    2016-01-01

    The regulation of L-type Ca2+ channels by protein kinase A (PKA) represents a crucial element within cardiac, skeletal muscle and neurological systems. Although much work has been done to understand this regulation in cardiac CaV1.2 Ca2+ channels, relatively little is known about the closely related CaV1.4 L-type Ca2+ channels, which feature prominently in the visual system. Here we find that CaV1.4 channels are indeed modulated by PKA phosphorylation within the inhibitor of Ca2+-dependent inactivation (ICDI) motif. Phosphorylation of this region promotes the occupancy of calmodulin on the channel, thus increasing channel open probability (PO) and Ca2+-dependent inactivation. Although this interaction seems specific to CaV1.4 channels, introduction of ICDI1.4 to CaV1.3 or CaV1.2 channels endows these channels with a form of PKA modulation, previously unobserved in heterologous systems. Thus, this mechanism may not only play an important role in the visual system but may be generalizable across the L-type channel family. PMID:27456671

  12. Protein kinase A modulation of CaV1.4 calcium channels

    NASA Astrophysics Data System (ADS)

    Sang, Lingjie; Dick, Ivy E.; Yue, David T.

    2016-07-01

    The regulation of L-type Ca2+ channels by protein kinase A (PKA) represents a crucial element within cardiac, skeletal muscle and neurological systems. Although much work has been done to understand this regulation in cardiac CaV1.2 Ca2+ channels, relatively little is known about the closely related CaV1.4 L-type Ca2+ channels, which feature prominently in the visual system. Here we find that CaV1.4 channels are indeed modulated by PKA phosphorylation within the inhibitor of Ca2+-dependent inactivation (ICDI) motif. Phosphorylation of this region promotes the occupancy of calmodulin on the channel, thus increasing channel open probability (PO) and Ca2+-dependent inactivation. Although this interaction seems specific to CaV1.4 channels, introduction of ICDI1.4 to CaV1.3 or CaV1.2 channels endows these channels with a form of PKA modulation, previously unobserved in heterologous systems. Thus, this mechanism may not only play an important role in the visual system but may be generalizable across the L-type channel family.

  13. Glucocorticoids and protein kinase A coordinately modulate transcription factor recruitment at a glucocorticoid-responsive unit.

    PubMed Central

    Espinás, M L; Roux, J; Pictet, R; Grange, T

    1995-01-01

    The rat tyrosine aminotransferase gene is a model system to study transcriptional regulation by glucocorticoid hormones. We analyzed transcription factor binding to the tyrosine aminotransferase gene glucocorticoid-responsive unit (GRU) at kb -2.5, using in vivo footprinting studies with both dimethyl sulfate and DNase I. At this GRU, glucocorticoid activation triggers a disruption of the nucleosomal structure. We show here that various regulatory pathways affect transcription factor binding to this GRU. The binding differs in two closely related glucocorticoid-responsive hepatoma cell lines. In line H4II, glucocorticoid induction promotes the recruitment of hepatocyte nuclear factor 3 (HNF3), presumably through the nucleosomal disruption. However, the footprint of the glucocorticoid receptor (GR) is not visible, even though a regular but transient interaction of the GR is necessary to maintain HNF3 binding. In contrast, in line FTO2B, HNF3 binds to the GRU in the absence of glucocorticoids and nucleosomal disruption, showing that a "closed" chromatin conformation does not repress the binding of certain transcription factors in a uniform manner. In FTO2B cells, the footprint of the GR is detectable, but this requires the activation of protein kinase A. In addition, protein kinase A stimulation also improves the recruitment of HNF3 independently of glucocorticoids and enhances the glucocorticoid response mediated by this GRU in an HNF3-dependent manner. In conclusion, the differences in the behavior of this regulatory sequence in the two cell lines show that various regulatory pathways are integrated at this GRU through modulation of interrelated events: transcription factor binding to DNA and nucleosomal disruption. PMID:7565684

  14. Antihypertensive action of 2-hydroxyoleic acid in SHRs via modulation of the protein kinase A pathway and Rho kinase.

    PubMed

    Alemany, Regina; Vögler, Oliver; Terés, Silvia; Egea, Carolina; Baamonde, Carmela; Barceló, Francisca; Delgado, Carlos; Jakobs, Karl H; Escribá, Pablo V

    2006-08-01

    Olive oil consumption leads to high monounsaturated fatty acid intake, especially oleic acid, and has been associated with a reduced risk of hypertension. However, the molecular mechanisms and contribution of its different components to lower blood pressure (BP) require further evaluation. Here, we examined whether a synthetic, non-beta-oxidation-metabolizable derivative of oleic acid, 2-hydroxyoleic acid (2-OHOA), can normalize BP in adult spontaneously hypertensive rats (SHRs) and whether its antihypertensive action involves cAMP-dependent protein kinase A (PKA) and Rho kinase, two major regulators of vascular smooth muscle contraction. Oral administration of 2-OHOA to SHRs induced sustained systolic BP decreases in a time-dependent (1-7 days) and dose-dependent (100-900 mg/kg every 12 h) manner. After 7 days of treatment with 2-OHOA (600 mg/kg), the systolic BP of SHRs was similar to that of normotensive Wistar Kyoto rats, returning to its initial hypertensive level after withdrawal of 2-OHOA. This treatment strongly increased the protein expression of the catalytic and regulatory RIalpha and RIIalpha PKA subunits as well as PKA activity in aortas from SHRs. Consistently, administration of the PKA inhibitor 8-bromo adenosine-3',5'-cyclic monophosphorothioate, Rp isomer, to 2-OHOA-treated SHRs induced a pronounced reversal (up to 59%) of the antihypertensive effect of 2-OHOA. Additionally, 2-OHOA completely reversed the pathological overexpression of aortic Rho kinase found in SHRs, suppressing the vasoconstrictory Rho kinase pathway.

  15. Bile acids modulate the Golgi membrane fission process via a protein kinase Ceta and protein kinase D-dependent pathway in colonic epithelial cells.

    PubMed

    Byrne, Anne-Marie; Foran, Eilis; Sharma, Ruchika; Davies, Anthony; Mahon, Ciara; O'Sullivan, Jacintha; O'Donoghue, Diarmuid; Kelleher, Dermot; Long, Aideen

    2010-04-01

    Deoxycholic acid (DCA) is a secondary bile acid that modulates signalling pathways in epithelial cells. DCA has been implicated in pathogenesis of colon carcinoma, particularly by activation of the protein kinase C (PKC) pathway. Ursodeoxycholic acid (UDCA), a tertiary bile acid, has been observed to have chemopreventive effects. The aim of this study was to investigate the effect of DCA and UDCA on the subcellular localization and activity of PKCeta and its downstream effects on Golgi structure in a colon cancer cell model. PKCeta expression was localized to the Golgi in HCT116 colon cancer cells. DCA induced fragmentation of the Golgi in these cells following activation of PKCeta and its downstream effector protein kinase D (PKD). Pretreatment of cells with UDCA or a glucocorticoid, dexamethasone, inhibited DCA-induced PKCeta/PKD activation and Golgi fragmentation. Knockdown of glucocorticoid receptor (GR) expression using small interfering RNA or inhibition using the GR antagonist mifepristone attenuated the inhibitory effect of UDCA on Golgi fragmentation. Elevated serum and faecal levels of DCA have been previously reported in patients with ulcerative colitis (UC) and colon cancer. Analysis of Golgi architecture in vivo using tissue microarrays revealed Golgi fragmentation in UC and colorectal cancer tissue. We have demonstrated that DCA can disrupt the structure of the Golgi, an organelle critical for normal cell function. Inhibition of this DCA-induced Golgi fragmentation by UDCA was mediated via the GR. This represents a potential mechanism of observed chemopreventive effects of UDCA in benign and malignant disease of the colon.

  16. Modulation of Brahma expression by the mitogen-activated protein kinase/extracellular signal regulated kinase pathway is associated with changes in melanoma proliferation.

    PubMed

    Mehrotra, Aanchal; Saladi, Srinivas Vinod; Trivedi, Archit R; Aras, Shweta; Qi, Huiling; Jayanthy, Ashika; Setaluri, Vijayasaradhi; de la Serna, Ivana L

    2014-12-01

    Brahma (BRM) and Brahma-related gene 1(BRG1) are catalytic subunits of SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes. BRM is epigenetically silenced in a wide-range of tumors. Mutations in the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene occur frequently in melanoma and lead to constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK1/2) pathway. We tested the hypothesis that BRM expression is modulated by oncogenic BRAF and phosphorylation of ERK1/2 in melanocytes and melanoma cells. Expression of oncogenic BRAF in melanocytes and melanoma cells that are wild-type for BRAF decreased BRM expression and increased BRG1 expression. Inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) or selective inhibition of BRAF in melanoma cells that harbor oncogenic BRAF increased BRM expression and decreased BRG1 expression. Increased BRM expression was associated with increased histone acetylation on the BRM promoter. Over-expression of BRM in melanoma cells that harbor oncogenic BRAF promoted changes in cell cycle progression and apoptosis consistent with a tumor suppressive role. Upon inhibition of BRAF(V600E) with PLX4032, BRM promoted survival. PLX4032 induced changes in BRM function were correlated with increased acetylation of the BRM protein. This study provides insights into the epigenetic consequences of inhibiting oncogenic BRAF in melanoma through modulation of SWI/SNF subunit expression and function.

  17. Modulation of influenza virus replication by alteration of sodium ion transport and protein kinase C activity

    PubMed Central

    Hoffmann, H.-Heinrich; Palese, Peter; Shaw, Megan L.

    2008-01-01

    In recent years, increasing levels of resistance to the four FDA-approved anti-influenza virus drugs have been described and vaccine manufacturers have experienced demands that exceed their capacity. This situation underlines the urgent need for novel antivirals as well as innovations in vaccine production in preparation for the next influenza epidemic. Here we report the development of a cell-based high-throughput screen which we have used for the identification of compounds that modulate influenza virus growth either negatively or positively. We screened a library of compounds with known biological activity and identified distinct groups of inhibitors and enhancers that target sodium channels or protein kinase C (PKC). We confirmed these results in viral growth assays and find that treatment with a sodium channel opener or PKC inhibitor significantly reduces viral replication. In contrast, inhibition of sodium channels or activation of PKC leads to enhanced virus production in tissue culture. These diametrically opposing effects strongly support a role for PKC activity and the regulation of Na+ currents in influenza virus replication and both may serve as targets for antiviral drugs. Furthermore, we raise the possibility that compounds that result in increased viral titers may be beneficial for boosting the production of tissue culture-grown influenza vaccines. PMID:18585796

  18. Aplotaxene blocks T cell activation by modulation of protein kinase C-θ-dependent pathway.

    PubMed

    Na, Bo-Ra; Kim, Hye-Ran; Kwon, Min-Sung; Lee, Hyun-Su; Piragyte, Indre; Choi, Eun-Ju; Choi, Hyun-Kyu; Han, Weon-Cheol; Lee, Seung-Ho; Jun, Chang-Duk

    2013-12-01

    Aplotaxene, (8Z, 11Z, 14Z)-heptadeca-1, 8, 11, 14-tetraene, is one of the major components of essential oil obtained from Inula helenium root, which is used in Oriental medicine. However, the effects of aplotaxene on immunity have not been investigated. Here, we show that aplotaxene inhibits T cell activation in terms of IL-2 and CD69 expression. Aplotaxene, at a concentration that optimally inhibits IL-2 production, has little effect on apoptotic or necrotic cell death, suggesting that apoptosis is not a mechanism for aplotaxene-mediated inhibition of T cell activation. Aplotaxene affects neither superantigeninduced conjugate formation between Jurkat T cells and Raji B cells nor clustering of CD3 and LFA-1 at the immunological synapse. Aplotaxene significantly inhibits PKC-θ phosphorylation and translocation to the immunological synapse, and blocks PMA-induced T-cell receptor internalization. Furthermore, aplotaxene leads to inhibition of mitogen-activated protein kinases (JNK, ERK and p38) phosphorylation and NF-κB, NF-AT, and AP-1 promoter activities in Jurkat T cells. Taken together, our findings provide evidence for the immunosuppressive effect of aplotaxene on activated T cells through the modulation of the PKC-θ and MAPK pathways, suggesting that aplotaxene may be a novel immunotherapeutic agent for immunological diseases related to the overactivation of T cells.

  19. Pb2+ via protein kinase C inhibits nicotinic cholinergic modulation of synaptic transmission in the hippocampus.

    PubMed

    Braga, Maria F M; Pereira, Edna F R; Mike, Arpad; Albuquerque, Edson X

    2004-11-01

    The present study was designed to investigate the effects of Pb(2+) on modulation of synaptic transmission by nicotinic receptors (nAChRs) in the rat hippocampus. To this end, inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs, respectively) were recorded by means of the whole-cell mode of the patch-clamp technique from rat hippocampal neurons in culture. Acetylcholine (ACh, 1 mM; 1-s pulses) triggered GABA release via activation of alpha4beta2* and alpha7* nAChRs. It also triggered glutamate release via activation of alpha7* nAChRs. Pb(2+) (0.1 and 1 microM) blocked ACh-triggered transmitter release. Blockade by Pb(2+) of ACh-triggered IPSCs was partially reversible upon washing of the neurons. In contrast, even after 30- to 60-min washing, there was no reversibility of Pb(2+)-induced blockade of ACh-triggered EPSCs. The effects of Pb(2+) on GABA release triggered by activation of alpha7* and alpha4beta2* nACRs were mimicked by the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (1 microM) and blocked by the indolocarbazole Go 7874 (50 nM) and the bisindolylmaleimide Ro-31-8425 (150 nM), which are selective PKC inhibitors. After washing of fully functional neuronal networks that had been exposed for 5 min to Pb(2+), the irreversible inhibition by Pb(2+) of ACh-triggered glutamate release was partially overridden by a disinhibitory mechanism that is likely to involve alpha4beta2* nAChR activation in interneurons that synapse onto other interneurons synapsing onto pyramidal neurons. Long-lasting inhibition of alpha7* nAChR modulation of synaptic transmission may contribute to the persistent cognitive impairment that results from childhood Pb(2+) intoxication.

  20. An immediate-early protein of white spot syndrome virus modulates the phosphorylation of focal adhesion kinase of shrimp.

    PubMed

    Lu, Huasong; Ruan, Lingwei; Xu, Xun

    2011-10-25

    WSSV interacts with integrin during infection of shrimps and modulate the focal adhesion kinase which is known as a regulator of several downstream signaling pathways. Viral protein kinases are thought to be important for virus infection by regulating the host signaling pathways. WSV083 is an immediate-early gene of white spot syndrome virus that contains a Ser/Thr protein kinase domain. So, does WSSV modulate FAK phosphorylation via the WSV083 molecule? In this study, co-transfection of WSV083 and MjFAK genes proceeded in insect cells revealed that the MjFAK phosphorylation and cell adhesion activity could be inhibited by the expression of WSV083. Kinase domain mutants of WSV083 lost its ability of inhibiting FAK phosphorylation. Moreover, silencing of FAK gene through RNAi accelerated the shrimp death rate upon WSSV challenge. These results demonstrate for the first time that modulation of FAK phosphorylation by WSV083 plays a critical role in the pathogenesis of WSSV infection.

  1. Protein KinaseModulates Ligand-induced Cell Surface Death Receptor Accumulation

    PubMed Central

    Meng, Xue Wei; Heldebrant, Michael P.; Flatten, Karen S.; Loegering, David A.; Dai, Haiming; Schneider, Paula A.; Gomez, Timothy S.; Peterson, Kevin L.; Trushin, Sergey A.; Hess, Allan D.; Smith, B. Douglas; Karp, Judith E.; Billadeau, Daniel D.; Kaufmann, Scott H.

    2010-01-01

    Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-α-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2–5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCα, PKCβ, PKCϵ, and PKCθ, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCβ shRNA uniquely reversed PMA-induced protection against cell death. The PKCβ-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCβ shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCβ, to tumor necrosis factor-α related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death. PMID:19887445

  2. Corosolic acid protects hepatocytes against ethanol-induced damage by modulating mitogen-activated protein kinases and activating autophagy.

    PubMed

    Guo, Xiaolan; Cui, Ruibing; Zhao, Jianjian; Mo, Rui; Peng, Lei; Yan, Ming

    2016-11-15

    The reactive oxygen species(ROS)/mitogen-activated protein kinase (MAPK) destroyed autophagy and the reactive oxygen species/mitogen-activated protein kinase (MAPK) pathway are considered closely related to ethanol-induced hepatocellular injury. Previous work indicated that corosolic acid, the natural extracts of leaves of the banaba tree, Lagerstroemia speciosa L., could protect the liver against ethanol-induced damage, but the underlying mechanism is unclear. In the study we found that corosolic acid significantly inhibited ethanol-induced apoptosis, increased level of tumor necrosis factor-α(TNF-α) and reactive oxygen species accumulation in vitro. Corosolic acid inhibited ethanol-activated p38 and c-Jun N-terminal kinase MAPK signaling in BRL-3A and HepG2 cells as well as in experimental rats. Corosolic acid restored the ethanol-suppressed expression of autophagy-related genes, including beclin-1 and the ratio of microtubule-associated protein light chain 3II/I (LC3II/I) via AMP-activated protein kinase (AMPK) activation both in vitro and in vivo. In experimental rats, corosolic acid ameliorated the detrimental histopathological findings. Corosolic acid may protect the liver against ethanol-induced injury by modulation of MAPK signaling and autophagy activation. These findings suggested that corosolic acid might be a promising agent in treatment of alcoholic liver diseases.

  3. Diacylglycerol kinase delta and protein kinase C(alpha) modulate epidermal growth factor receptor abundance and degradation through ubiquitin-specific protease 8.

    PubMed

    Cai, Jinjin; Crotty, Tracy M; Reichert, Ethan; Carraway, Kermit L; Stafforini, Diana M; Topham, Matthew K

    2010-03-05

    Many human epithelial cancers are characterized by abnormal activation of the epidermal growth factor receptor (EGFR), which is often caused by its excessive expression in tumor cells. The abundance of EGFR is modulated, in part, by its ubiquitination, which targets it for degradation. The components responsible for adding ubiquitin to EGFR are well characterized, but this is a reversible process, and the mechanisms that modulate the removal of ubiquitin from the EGFR are not well known. We found that de-ubiquitination of EGFR was regulated by diacylglycerol kinase delta (DGKdelta), a lipid kinase that terminates diacylglycerol signaling. In DGKdelta-deficient cells, ubiquitination of EGFR was enhanced, which attenuated the steady-state levels of EGFR and promoted its ligand-induced degradation. These effects were not caused by changes in the ubiquitinating apparatus, but instead were due to reduced expression of the de-ubiquitinase, ubiquitin-specific protease 8 (USP8). Depletion of protein kinase Calpha (PKCalpha), a target of diacylglycerol, rescued the levels of USP8 and normalized EGFR degradation in DGKdelta-deficient cells. Moreover, the effects of PKCalpha were caused by its inhibition of Akt, which stabilizes USP8. Our data indicate a novel mechanism where DGKdelta and PKCalpha modulate the levels of ubiquitinated EGFR through Akt and USP8.

  4. AIK1, A Mitogen-Activated Protein Kinase, Modulates Abscisic Acid Responses through the MKK5-MPK6 Kinase Cascade1[OPEN

    PubMed Central

    Li, Kun; Yang, Fengbo; Zhang, Guozeng; Song, Shufei; Li, Yuan; Ren, Dongtao; Miao, Yuchen

    2017-01-01

    The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily conserved signal transduction module involved in transducing extracellular signals to the nucleus for appropriate cellular adjustment. This cascade essentially consists of three components: a MAPK kinase kinase (MAPKKK), a MAPK kinase, and a MAPK, connected to each other by the event of phosphorylation. Here, we report the characterization of a MAPKKK, ABA-INSENSITIVE PROTEIN KINASE1 (AIK1), which regulates abscisic acid (ABA) responses in Arabidopsis (Arabidopsis thaliana). T-DNA insertion mutants of AIK1 showed insensitivity to ABA in terms of both root growth and stomatal response. AIK1 functions in ABA responses via regulation of root cell division and elongation, as well as stomatal responses. The activity of AIK1 is induced by ABA in Arabidopsis and tobacco (Nicotiana benthamiana), and the Arabidopsis protein phosphatase type 2C, ABI1, a negative regulator of ABA signaling, restricts AIK1 activity by dephosphorylation. Bimolecular fluorescence complementation analysis showed that MPK3, MPK6, and AIK1 interact with MKK5. The single mutant seedlings of mpk6 and mkk5 have similar phenotypes to aik1, but mkk4 does not. AIK1 was localized in the cytoplasm and shown to activate MKK5 by protein phosphorylation, which was an ABA-activated process. Constitutively active MKK5 in aik1 mutant seedlings complements the ABA-insensitive root growth phenotype of aik1. The activity of MPK6 was increased by ABA in wild-type seedlings, but its activation by ABA was impaired in aik1 and aik1 mkk5 mutants. These findings clearly suggest that the AIK1-MKK5-MPK6 cascade functions in the ABA regulation of primary root growth and stomatal response. PMID:27913741

  5. Microbiota Modulates Behavior and Protein Kinase C mediated cAMP response element-binding protein Signaling

    PubMed Central

    Zeng, Li; Zeng, Benhua; Wang, Haiyang; Li, Bo; Huo, Ran; Zheng, Peng; Zhang, Xiaotong; Du, Xiangyu; Liu, Meiling; Fang, Zheng; Xu, Xuejiao; Zhou, Chanjuan; Chen, Jianjun; Li, Wenxia; Guo, Jing; Wei, Hong; Xie, Peng

    2016-01-01

    Evolutionary pressure drives gut microbiota–host coevolution and results in complex interactions between gut microbiota and neural development; however, the molecular mechanisms by which the microbiota governs host behavior remain obscure. Here, we report that colonization early in life is crucial for the microbiota to modulate brain development and behavior; later colonization or deletion of microbiota cannot completely reverse the behaviors. Microarray analysis revealed an association between absence of gut microbiota and expression in cAMP responding element-binding protein (CREB) regulated genes in the hippocampus. The absence of gut microbiota from birth was shown to be associated with decreased CREB expression, followed by decreases of protein kinase C beta (PRKCB) and AMPA receptors expression, and an increase of phosphorylation CREB (pCREB) expression. Microbiota colonization in adolescence restored CREB and pCREB expression, but did not alter PRKCB and AMPARs expression. The removal of the gut microbiota from SPF mice using antibiotics only reduced pCREB expression. These findings suggest that (i) colonization of the gut microbiota early in life might facilitate neurodevelopment via PKC–CREB signaling and (ii) although GF mice and ABX mice display reduced anxiety-related behaviors, the molecular mechanisms behind this might differ. PMID:27444685

  6. Redox Regulation of Protein Kinases

    PubMed Central

    Truong, Thu H.; Carroll, Kate S.

    2015-01-01

    Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous H2O2 by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiologic and pathological H2O2 responses. PMID:23639002

  7. Differential Role of β1C and β1A Integrin Cytoplasmic Variants in Modulating Focal Adhesion Kinase, Protein Kinase B/AKT, and Ras/Mitogen-activated Protein Kinase Pathways

    PubMed Central

    Fornaro, Mara; Steger, Craig A.; Bennett, Anton M.; Wu, J. Julie; Languino, Lucia R.

    2000-01-01

    The integrin cytoplasmic domain modulates cell proliferation, adhesion, migration, and intracellular signaling. The β1 integrin subunits, β1C and β1A, that contain variant cytoplasmic domains differentially affect cell proliferation; β1C inhibits proliferation, whereas β1A promotes it. We investigated the ability of β1C and β1A to modulate integrin-mediated signaling events that affect cell proliferation and survival in Chinese hamster ovary stable cell lines expressing either human β1C or human β1A. The different cytodomains of either β1C or β1A did not affect either association with the endogenous α2, αV, and α5 subunits or cell adhesion to fibronectin or TS2/16, a mAb to human β1. Upon engagement of endogenous and exogenous integrins by fibronectin, cells expressing β1C showed significantly inhibited extracellular signal–regulated kinase (ERK) 2 activation compared with β1A stable cell lines. In contrast, focal adhesion kinase phosphorylation and Protein Kinase B/AKT activity were not affected. Selective engagement of the exogenously expressed β1C by TS2/16 led to stimulation of Protein Kinase B/AKT phosphorylation but not of ERK2 activation; in contrast, β1A engagement induced activation of both proteins. We show that Ras activation was strongly reduced in β1C stable cell lines in response to fibronectin adhesion and that expression of constitutively active Ras, Ras 61 (L), rescued β1C-mediated down-regulation of ERK2 activation. Inhibition of cell proliferation in β1C stable cell lines was attributable to an inhibitory effect of β1C on the Ras/MAP kinase pathway because expression of activated MAPK kinase rescued β1C antiproliferative effect. These findings show that the β1C variant, by means of a unique signaling mechanism, selectively inhibits the MAP kinase pathway by preventing Ras activation without affecting either survival signals stimulated by integrins or cellular interactions with the extracellular matrix. These findings

  8. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  9. Death-associated protein kinase-mediated cell death modulated by interaction with DANGER.

    PubMed

    Kang, Bingnan N; Ahmad, Abdullah S; Saleem, Sofiyan; Patterson, Randen L; Hester, Lynda; Doré, Sylvain; Snyder, Solomon H

    2010-01-06

    Death-associated protein kinase (DAPK) is a key player in multiple cell death signaling pathways. We report that DAPK is regulated by DANGER, a partial MAB-21 domain-containing protein. DANGER binds directly to DAPK and inhibits DAPK catalytic activity. DANGER-deficient mouse embryonic fibroblasts and neurons exhibit greater DAPK activity and increased sensitivity to cell death stimuli than do wild-type control cells. In addition, DANGER-deficient mice manifest more severe brain damage after acute excitotoxicity and transient cerebral ischemia than do control mice. Accordingly, DANGER may physiologically regulate the viability of neurons and represent a potential therapeutic target for stroke and neurodegenerative diseases.

  10. AMP-activated Protein Kinase Signaling Activation by Resveratrol Modulates Amyloid-β Peptide Metabolism*

    PubMed Central

    Vingtdeux, Valérie; Giliberto, Luca; Zhao, Haitian; Chandakkar, Pallavi; Wu, Qingli; Simon, James E.; Janle, Elsa M.; Lobo, Jessica; Ferruzzi, Mario G.; Davies, Peter; Marambaud, Philippe

    2010-01-01

    Alzheimer disease is an age-related neurodegenerative disorder characterized by amyloid-β (Aβ) peptide deposition into cerebral amyloid plaques. The natural polyphenol resveratrol promotes anti-aging pathways via the activation of several metabolic sensors, including the AMP-activated protein kinase (AMPK). Resveratrol also lowers Aβ levels in cell lines; however, the underlying mechanism responsible for this effect is largely unknown. Moreover, the bioavailability of resveratrol in the brain remains uncertain. Here we show that AMPK signaling controls Aβ metabolism and mediates the anti-amyloidogenic effect of resveratrol in non-neuronal and neuronal cells, including in mouse primary neurons. Resveratrol increased cytosolic calcium levels and promoted AMPK activation by the calcium/calmodulin-dependent protein kinase kinase-β. Direct pharmacological and genetic activation of AMPK lowered extracellular Aβ accumulation, whereas AMPK inhibition reduced the effect of resveratrol on Aβ levels. Furthermore, resveratrol inhibited the AMPK target mTOR (mammalian target of rapamycin) to trigger autophagy and lysosomal degradation of Aβ. Finally, orally administered resveratrol in mice was detected in the brain where it activated AMPK and reduced cerebral Aβ levels and deposition in the cortex. These data suggest that resveratrol and pharmacological activation of AMPK have therapeutic potential against Alzheimer disease. PMID:20080969

  11. Teaching resources. Protein kinases.

    PubMed

    Caplan, Avrom

    2005-02-22

    This Teaching Resource provides lecture notes and slides for a class covering the structure and function of protein kinases and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the genomics and evolutionary relationships among kinases and then proceeds to describe the structure-function relationships of specific kinases, the molecular mechanisms underlying substrate specificity, and selected issues in regulation of kinase activity.

  12. Protein Kinase C Inhibitors as Modulators of Vascular Function and Their Application in Vascular Disease

    PubMed Central

    Khalil, Raouf A.

    2013-01-01

    Blood pressure (BP) is regulated by multiple neuronal, hormonal, renal and vascular control mechanisms. Changes in signaling mechanisms in the endothelium, vascular smooth muscle (VSM) and extracellular matrix cause alterations in vascular tone and blood vessel remodeling and may lead to persistent increases in vascular resistance and hypertension (HTN). In VSM, activation of surface receptors by vasoconstrictor stimuli causes an increase in intracellular free Ca2+ concentration ([Ca2+]i), which forms a complex with calmodulin, activates myosin light chain (MLC) kinase and leads to MLC phosphorylation, actin-myosin interaction and VSM contraction. Vasoconstrictor agonists could also increase the production of diacylglycerol which activates protein kinase C (PKC). PKC is a family of Ca2+-dependent and Ca2+-independent isozymes that have different distributions in various blood vessels, and undergo translocation from the cytosol to the plasma membrane, cytoskeleton or the nucleus during cell activation. In VSM, PKC translocation to the cell surface may trigger a cascade of biochemical events leading to activation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK), a pathway that ultimately increases the myofilament force sensitivity to [Ca2+]i, and enhances actin-myosin interaction and VSM contraction. PKC translocation to the nucleus may induce transactivation of various genes and promote VSM growth and proliferation. PKC could also affect endothelium-derived relaxing and contracting factors as well as matrix metalloproteinases (MMPs) in the extracellular matrix further affecting vascular reactivity and remodeling. In addition to vasoactive factors, reactive oxygen species, inflammatory cytokines and other metabolic factors could affect PKC activity. Increased PKC expression and activity have been observed in vascular disease and in certain forms of experimental and human HTN. Targeting of vascular PKC using PKC inhibitors may function in concert with

  13. Mitogen- and stress-activated protein kinase 1 modulates photic entrainment of the suprachiasmatic circadian clock.

    PubMed

    Cao, Ruifeng; Butcher, Greg Q; Karelina, Kate; Arthur, J Simon; Obrietan, Karl

    2013-01-01

    The master circadian clock in mammals, the suprachiasmatic nucleus (SCN), is under the entraining influence of the external light cycle. At a mechanistic level, intracellular signaling via the p42/44 mitogen-activated protein kinase pathway appears to play a central role in light-evoked clock entrainment; however, the precise downstream mechanisms by which this pathway influences clock timing are not known. Within this context, we have previously reported that light stimulates activation of the mitogen-activated protein kinase effector mitogen-stress-activated kinase 1 (MSK1) in the SCN. In this study, we utilised MSK1(-/-) mice to further investigate the potential role of MSK1 in circadian clock timing and entrainment. Locomotor activity analysis revealed that MSK1 null mice entrained to a 12 h light/dark cycle and exhibited circadian free-running rhythms in constant darkness. Interestingly, the free-running period in MSK1 null mice was significantly longer than in wild-type control animals, and MSK1 null mice exhibited a significantly greater variance in activity onset. Further, MSK1 null mice exhibited a significant reduction in the phase-delaying response to an early night light pulse (100 lux, 15 min), and, using an 8 h phase-advancing 'jet-lag' experimental paradigm, MSK1 knockout animals exhibited a significantly delayed rate of re-entrainment. At the molecular level, early night light-evoked cAMP response element-binding protein (CREB) phosphorylation, histone phosphorylation and Period1 gene expression were markedly attenuated in MSK1(-/-) animals relative to wild-type mice. Together, these data provide key new insights into the molecular mechanisms by which MSK1 affects the SCN clock.

  14. Structural properties of UMP-kinase from Escherichia coli: modulation of protein solubility by pH and UTP.

    PubMed

    Serina, L; Bucurenci, N; Gilles, A M; Surewicz, W K; Fabian, H; Mantsch, H H; Takahashi, M; Petrescu, I; Batelier, G; Bârzu, O

    1996-06-04

    UMP-kinase from Escherichia coli, unlike the analogous enzyme from eukaryotic organisms, is an oligomeric protein subjected to complex regulatory mechanisms in which UTP and GTP act as allosteric effectors. While the enzyme has an unusually low solubility at neutral pH (< or = 0.1 mg of protein/ mL), its solubility increases markedly above pH 8 and below pH 4. Furthermore, the solubility of the bacterial UMP-kinase at neutral pH is greatly enhanced in the presence of Mg-free UTP. Thermal denaturation experiments have demonstrated that UTP also increases the stability of the protein. Fourier-transform infrared spectroscopy and circular dichroism show that the secondary structure of the protein is the same at neutral and at alkaline pH. These data indicate that variations in enzyme solubility must be related to subtle changes in the tertiary and/or quaternary structure which modulate the exposure of hydrophobic surfaces in the protein molecule. A variant of UMP-kinase, obtained by site-directed mutagenesis (Asp159Asn), which is similar to the wild-type enzyme in its stability and kinetic properties, has a much increased water solubility (> 5 mg protein/mL) even at neutral pH. This suggests that salt bridges may be involved in the equilibrium between the soluble and aggregated forms of the wild-type enzyme, and that conformational changes induced upon binding of UTP increase the protein solubility by disrupting these salt bridges.

  15. The mitogen-activated protein kinase Slt2 modulates arsenite transport through the aquaglyceroporin Fps1.

    PubMed

    Ahmadpour, Doryaneh; Maciaszczyk-Dziubinska, Ewa; Babazadeh, Roja; Dahal, Sita; Migocka, Magdalena; Andersson, Mikael; Wysocki, Robert; Tamás, Markus J; Hohmann, Stefan

    2016-10-01

    Arsenite is widely present in nature; therefore, cells have evolved mechanisms to prevent arsenite influx and promote efflux. In yeast (Saccharomyces cerevisiae), the aquaglyceroporin Fps1 mediates arsenite influx and efflux. The mitogen-activated protein kinase (MAPK) Hog1 has previously been shown to restrict arsenite influx through Fps1. In this study, we show that another MAPK, Slt2, is transiently phosphorylated in response to arsenite influx. Our findings indicate that the protein kinase activity of Slt2 is required for its role in arsenite tolerance. While Hog1 prevents arsenite influx via phosphorylation of T231 at the N-terminal domain of Fps1, Slt2 promotes arsenite efflux through phosphorylation of S537 at the C terminus. Our data suggest that Slt2 physically interacts with Fps1 and that this interaction depends on phosphorylation of S537. We hypothesize that Hog1 and Slt2 may affect each other's binding to Fps1, thereby controlling the opening and closing of the channel.

  16. Protein interaction module-assisted function X (PIMAX) approach to producing challenging proteins including hyperphosphorylated tau and active CDK5/p25 kinase complex.

    PubMed

    Sui, Dexin; Xu, Xinjing; Ye, Xuemei; Liu, Mengyu; Mianecki, Maxwell; Rattanasinchai, Chotirat; Buehl, Christopher; Deng, Xiexiong; Kuo, Min-Hao

    2015-01-01

    Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator," such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and application of a protein interaction module-assisted function X (PIMAX) system that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of an enzymatically active cyclin-dependent kinase complex and hyperphosphorylated tau protein, which is intimately linked to Alzheimer disease. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3β and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad implications for proteomics and biomedical research.

  17. A calcium sensor – protein kinase signaling module diversified in plants and is retained in all lineages of Bikonta species

    PubMed Central

    Beckmann, Linda; Edel, Kai H.; Batistič, Oliver; Kudla, Jörg

    2016-01-01

    Calcium (Ca2+) signaling is a universal mechanism of signal transduction and involves Ca2+ signal formation and decoding of information by Ca2+ binding proteins. Calcineurin B-like proteins (CBLs), which upon Ca2+ binding activate CBL-interacting protein kinases (CIPKs) regulate a multitude of physiological processes in plants. Here, we combine phylogenomics and functional analyses to investigate the occurrence and structural conservation of CBL and CIPK proteins in 26 species representing all major clades of eukaryotes. We demonstrate the presence of at least singular CBL-CIPK pairs in representatives of Archaeplastida, Chromalveolates and Excavates and their general absence in Opisthokonta and Amoebozoa. This denotes CBL-CIPK complexes as evolutionary ancient Ca2+ signaling modules that likely evolved in the ancestor of all Bikonta. Furthermore, we functionally characterize the CBLs and CIPK from the parabasalid human pathogen Trichomonas vaginalis. Our results reveal strict evolutionary conservation of functionally important structural features, preservation of biochemical properties and a remarkable cross-kingdom protein-protein interaction potential between CBLs and CIPKs from Arabidopsis thaliana and T. vaginalis. Together our findings suggest an ancient evolutionary origin of a functional CBL-CIPK signaling module close to the root of eukaryotic evolution and provide insights into the initial evolution of signaling networks and Ca2+ signaling specificity. PMID:27538881

  18. Modulation of cytoskeletal dynamics by mammalian nucleoside diphosphate kinase (NDPK) proteins.

    PubMed

    Snider, Natasha T; Altshuler, Peter J; Omary, M Bishr

    2015-02-01

    Nucleoside diphosphate kinase (NDPK) proteins comprise a family of ten human isoforms that participate in the regulation of multiple cellular processes via enzymatic and nonenzymatic functions. The major enzymatic function of NDPKs is the generation of nucleoside triphosphates, such as guanosine triphosphate (GTP). Mechanisms behind the nonenzymatic NDPK functions are not clear but likely involve context-dependent signaling roles of NDPK within multi-protein complexes. This is most evident for NDPK-A, which is encoded by the human NME1 gene, the first tumor metastasis suppressor gene to be identified. Understanding which protein interactions are most relevant for the biological and metastasis-related functions of NDPK will be important in the potential utilization of NDPK as a disease target. Accumulating evidence suggests that NDPK interacts with and affects various components and regulators of the cytoskeleton, including actin-binding proteins, intermediate filaments, and cytoskeletal attachment structures (adherens junctions, desmosomes, and focal adhesions). We review the existing literature on this topic and highlight outstanding questions and potential future directions that should clarify the impact of NDPK on the different cytoskeletal systems.

  19. Manganese modulation of MAPK pathways: effects on upstream mitogen activated protein kinase kinases (MKKs) and mitogen activated kinase phosphatase-1 (MKP-1) in microglial cells

    PubMed Central

    Crittenden, Patrick L.; Filipov, Nikolay M.

    2010-01-01

    Multiple studies demonstrate that manganese (Mn) exposure potentiates inflammatory mediator output from activated glia; this increased output is associated with enhanced mitogen activated protein kinase (MAPK: p38, ERK, and JNK) activity. We hypothesized that Mn activates MAPK by activating the kinases upstream of MAPK, i.e., MKK-3/6, MKK-1/2, and MKK-4 (responsible for activation of p38, ERK, and JNK, respectively), and/or by inhibiting a major phosphatase responsible for MAPK inactivation, MKP-1. Exposure of N9 microglia to Mn (250μM), LPS (100 ng/ml), or Mn+LPS increased MKK-3/6 and MKK-4 activity at 1 h; the effect of Mn+LPS on MKK-4 activation was greater than the rest. At 4 h, Mn, LPS, and Mn+LPS increased MKK-3/6 and MKK-1/2 phosphorylation, whereas MKK-4 was activated only by Mn and Mn+LPS. Besides activating MKK-4 via Ser257/Thr261 phosphorylation, Mn (4 h) prevented MKK-4’s phosphorylation on Ser80, which negatively regulates MKK-4 activity. Exposure to Mn or Mn+LPS (1 h) decreased both mRNA and protein expression of MKP-1, the negative MAPK regulator. In addition, we observed that at 4 h, but not at 1 h, a time point coinciding with increased MAPK activity, Mn+LPS markedly increased TNF-α , IL-6, and Cox-2 mRNA, suggesting a delayed effect. The fact that all three major groups of MKKs, MKK-1/2, MKK-3/6, and MKK-4 are activated by Mn suggests that Mn-induced activation of MAPK occurs via traditional mechanisms, which perhaps involve the MAPKs farthest upstream, MKKKs (MAP3Ks). In addition, for all MKKs, Mn-induced activation was persistent at least for 4 h, indicating a long-term effect. PMID:20589745

  20. Protein kinase A mediates adenosine A2a receptor modulation of neurotransmitter release via synapsin I phosphorylation in cultured cells from medulla oblongata.

    PubMed

    Matsumoto, Joao Paulo Pontes; Almeida, Marina Gomes; Castilho-Martins, Emerson Augusto; Costa, Maisa Aparecida; Fior-Chadi, Debora Rejane

    2014-08-01

    Synaptic transmission is an essential process for neuron physiology. Such process is enabled in part due to modulation of neurotransmitter release. Adenosine is a synaptic modulator of neurotransmitter release in the Central Nervous System, including neurons of medulla oblongata, where several nuclei are involved with neurovegetative reflexes. Adenosine modulates different neurotransmitter systems in medulla oblongata, specially glutamate and noradrenaline in the nucleus tractussolitarii, which are involved in hypotensive responses. However, the intracellular mechanisms involved in this modulation remain unknown. The adenosine A2a receptor modulates neurotransmitter release by activating two cAMP protein effectors, the protein kinase A and the exchange protein activated by cAMP. Therefore, an in vitro approach (cultured cells) was carried out to evaluate modulation of neurotransmission by adenosine A2a receptor and the signaling intracellular pathway involved. Results show that the adenosine A2a receptor agonist, CGS 21680, increases neurotransmitter release, in particular, glutamate and noradrenaline and such response is mediated by protein kinase A activation, which in turn increased synapsin I phosphorylation. This suggests a mechanism of A2aR modulation of neurotransmitter release in cultured cells from medulla oblongata of Wistar rats and suggest that protein kinase A mediates this modulation of neurotransmitter release via synapsin I phosphorylation.

  1. Aurora B kinase and protein phosphatase 1 have opposing roles in modulating kinetochore assembly.

    PubMed

    Emanuele, Michael J; Lan, Weijie; Jwa, Miri; Miller, Stephanie A; Chan, Clarence S M; Stukenberg, P Todd

    2008-04-21

    The outer kinetochore binds microtubules to control chromosome movement. Outer kinetochore assembly is restricted to mitosis, whereas the inner kinetochore remains tethered to centromeres throughout the cell cycle. The cues that regulate this transient assembly are unknown. We find that inhibition of Aurora B kinase significantly reduces outer kinetochore assembly in Xenopus laevis and human tissue culture cells, frog egg extracts, and budding yeast. In X. leavis M phase extracts, preassembled kinetochores disassemble after inhibiting Aurora B activity with either drugs or antibodies. Kinetochore disassembly, induced by Aurora B inhibition, is rescued by restraining protein phosphatase 1 (PP1) activity. PP1 is necessary for kinetochores to disassemble at the exit from M phase, and purified enzyme is sufficient to cause disassembly on isolated mitotic nuclei. These data demonstrate that Aurora B activity is required for kinetochore maintenance and that PP1 is necessary and sufficient to disassemble kinetochores. We suggest that Aurora B and PP1 coordinate cell cycle-dependent changes in kinetochore assembly though phosphorylation of kinetochore substrates.

  2. The Parkinson’s Disease-Associated Protein Kinase LRRK2 Modulates Notch Signaling through the Endosomal Pathway

    PubMed Central

    Imai, Yuzuru; Kobayashi, Yoshito; Inoshita, Tsuyoshi; Meng, Hongrui; Arano, Taku; Uemura, Kengo; Asano, Takeshi; Yoshimi, Kenji; Zhang, Chang-Liang; Matsumoto, Gen; Ohtsuka, Toshiyuki; Kageyama, Ryoichiro; Kiyonari, Hiroshi; Shioi, Go; Nukina, Nobuyuki; Hattori, Nobutaka; Takahashi, Ryosuke

    2015-01-01

    Leucine-rich repeat kinase 2 (LRRK2) is a key molecule in the pathogenesis of familial and idiopathic Parkinson’s disease (PD). We have identified two novel LRRK2-associated proteins, a HECT-type ubiquitin ligase, HERC2, and an adaptor-like protein with six repeated Neuralized domains, NEURL4. LRRK2 binds to NEURL4 and HERC2 via the LRRK2 Ras of complex proteins (ROC) domain and NEURL4, respectively. HERC2 and NEURL4 link LRRK2 to the cellular vesicle transport pathway and Notch signaling, through which the LRRK2 complex promotes the recycling of the Notch ligand Delta-like 1 (Dll1)/Delta (Dl) through the modulation of endosomal trafficking. This process negatively regulates Notch signaling through cis-inhibition by stabilizing Dll1/Dl, which accelerates neural stem cell differentiation and modulates the function and survival of differentiated dopaminergic neurons. These effects are strengthened by the R1441G ROC domain-mutant of LRRK2. These findings suggest that the alteration of Notch signaling in mature neurons is a component of PD etiology linked to LRRK2. PMID:26355680

  3. Protein kinase C promotes cardiac fibrosis and heart failure by modulating galectin-3 expression.

    PubMed

    Song, Xiang; Qian, Xiaoqian; Shen, Ming; Jiang, Rong; Wagner, Mary B; Ding, Guoliang; Chen, Guangping; Shen, Baozhong

    2015-02-01

    Protein kinase C (PKC) and galectin-3 are two important mediators that play a key pathogenic role in cardiac hypertrophy and heart failure (HF). However, the molecular mechanisms and signaling pathways are not fully understood. In this study, we explored the relationship between and roles of PKC-α and galectin-3 in the development of HF. We found that activation of PKC by phorbol dibutyrate (PDB) increased galectin-3 expression by ~180%, as well as collagen I and fibronection accumulation in cultured HL-1 cardiomyocytes. Over-expression of galectin-3 in HL-1 cells increased collagen I protein production. Inhibition of galectin-3 by β-lactose blocked PDB-induced galectin-3 and collagen production, indicating that galectin-3 mediates PKC-induced cardiac fibrosis. In rats subjected to pulmonary artery banding (PAB) to induce right ventricular HF, galectin-3 was increased by ~140% in the right ventricle and also by ~240% in left ventricle compared to control. The elevated galectin-3 is consistent with an increase of total and activated (phosphorylated) PKC-α, α-SMA and collagen I. Finally, we extended our findings to examine the role of angiotensin II (Ang II), which activates the PKC pathway and contributes to cardiac fibrosis and the development of HF. We found that Ang II activated the PKC-α pathway and increased galectin-3 expression and collagen production. This study provides a new insight into the molecular mechanisms of HF mediated by PKC-α and galectin-3. PKC-α promotes cardiac fibrosis and HF by stimulation of galectin-3 expression.

  4. Roles of Protein Kinase A and Adenylate Cyclase in Light-Modulated Cellulase Regulation in Trichoderma reesei

    PubMed Central

    Schuster, André; Tisch, Doris; Seidl-Seiboth, Verena; Kubicek, Christian P.

    2012-01-01

    The cyclic AMP (cAMP) pathway represents a central signaling cascade with crucial functions in all organisms. Previous studies of Trichoderma reesei (anamorph of Hypocrea jecorina) suggested a function of cAMP signaling in regulation of cellulase gene expression. We were therefore interested in how the crucial components of this pathway, adenylate cyclase (ACY1) and cAMP-dependent protein kinase A (PKA), would affect cellulase gene expression. We found that both ACY1 and PKA catalytic subunit 1 (PKAC1) are involved in regulation of vegetative growth but are not essential for sexual development. Interestingly, our results showed considerably increased transcript abundance of cellulase genes in darkness compared to light (light responsiveness) upon growth on lactose. This effect is strongly enhanced in mutant strains lacking PKAC1 or ACY1. Comparison to the wild type showed that ACY1 has a consistently positive effect on cellulase gene expression in light and darkness, while PKAC1 influences transcript levels of cellulase genes positively in light but negatively in darkness. A function of PKAC1 in light-modulated cellulase gene regulation is also reflected by altered complex formation within the cel6a/cbh2 promoter in light and darkness and in the absence of pkac1. Analysis of transcript levels of cellulase regulator genes indicates that the regulatory output of the cAMP pathway may be established via adjustment of XYR1 abundance. Consequently, both adenylate cyclase and protein kinase A are involved in light-modulated cellulase gene expression in T. reesei and have a dampening effect on the light responsiveness of this process. PMID:22286997

  5. Ceramides modulate protein kinase C activity and perturb the structure of Phosphatidylcholine/Phosphatidylserine bilayers.

    PubMed Central

    Huang, H W; Goldberg, E M; Zidovetzki, R

    1999-01-01

    We studied the effects of natural ceramide and a series of ceramide analogs with different acyl chain lengths on the activity of rat brain protein kinase C (PKC) and on the structure of bovine liver phosphatidylcholine (BLPC)/dipalmitoylphosphatidylcholine (DPPC)/dipalmitoylphosphatidylserine (DPPS) (3:1:1 molar ratio) bilayers using (2)H-NMR and specific enzymatic assays in the absence or presence of 7.5 mol % diolein (DO). Only a slight activation of PKC was observed upon addition of the short-chain ceramide analogs (C(2)-, C(6)-, or C(8)-ceramide); natural ceramide or C(16)-ceramide had no effect. In the presence of 7.5 mol % DO, natural ceramide and C(16)-ceramide analog slightly attenuated DO-enhanced PKC activity. (2)H-NMR results demonstrated that natural ceramide and C(16)-ceramide induced lateral phase separation of gel-like and liquid crystalline domains in the bilayers; however, this type of membrane perturbation has no direct effect on PKC activity. The addition of both short-chain ceramide analogs and DO had a synergistic effect in activating PKC, with maximum activity observed with 20 mol % C(6)-ceramide and 15 mol % DO. Further increases in C(6)-ceramide and/or DO concentrations led to decreased PKC activity. A detailed (2)H-NMR investigation of the combined effects of C(6)-ceramide and DO on lipid bilayer structure showed a synergistic effect of these two reagents to increase membrane tendency to adopt nonbilayer structures, resulting in the actual presence of such structures in samples exceeding 20 mol % ceramide and 15 mol % DO. Thus, the increased tendency to form nonbilayer lipid phases correlates with increased PKC activity, whereas the actual presence of such phases reduced the activity of the enzyme. Moreover, the results show that short-chain ceramide analogs, widely used to study cellular effects of ceramide, have biological effects that are not exhibited by natural ceramide. PMID:10465759

  6. Conserved herpesvirus protein kinases

    PubMed Central

    Gershburg, Edward; Pagano, Joseph S.

    2008-01-01

    Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein-Barr Virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task. PMID:17881303

  7. E6 variants of human papillomavirus 18 differentially modulate the protein kinase B/phosphatidylinositol 3-kinase (akt/PI3K) signaling pathway

    SciTech Connect

    Contreras-Paredes, Adriana

    2009-01-05

    Intra-type genome variations of high risk Human papillomavirus (HPV) have been associated with a differential threat for cervical cancer development. In this work, the effect of HPV18 E6 isolates in Akt/PKB and Mitogen-associated protein kinase (MAPKs) signaling pathways and its implication in cell proliferation were analyzed. E6 from HPV types 16 and 18 are able to bind and promote degradation of Human disc large (hDlg). Our results show that E6 variants differentially modulate hDlg degradation, rebounding in levels of activated PTEN and PKB. HPV18 E6 variants are also able to upregulate phospho-PI3K protein, strongly correlating with activated MAPKs and cell proliferation. Data was supported by the effect of E6 silencing in HPV18-containing HeLa cells, as well as hDlg silencing in the tested cells. Results suggest that HPV18 intra-type variations may derive in differential abilities to activate cell-signaling pathways such as Akt/PKB and MAPKs, directly involved in cell survival and proliferation.

  8. Regulation of glycogen synthesis by the laforin-malin complex is modulated by the AMP-activated protein kinase pathway.

    PubMed

    Solaz-Fuster, Maria Carmen; Gimeno-Alcañiz, José Vicente; Ros, Susana; Fernandez-Sanchez, Maria Elena; Garcia-Fojeda, Belen; Criado Garcia, Olga; Vilchez, David; Dominguez, Jorge; Garcia-Rocha, Mar; Sanchez-Piris, Maribel; Aguado, Carmen; Knecht, Erwin; Serratosa, Jose; Guinovart, Joan Josep; Sanz, Pascual; Rodriguez de Córdoba, Santiago

    2008-03-01

    Lafora progressive myoclonus epilepsy (LD) is a fatal autosomal recessive neurodegenerative disorder characterized by the presence of glycogen-like intracellular inclusions called Lafora bodies. LD is caused by mutations in two genes, EPM2A and EPM2B, encoding respectively laforin, a dual-specificity protein phosphatase, and malin, an E3 ubiquitin ligase. Previously, we and others have suggested that the interactions between laforin and PTG (a regulatory subunit of type 1 protein phosphatase) and between laforin and malin are critical in the pathogenesis of LD. Here, we show that the laforin-malin complex downregulates PTG-induced glycogen synthesis in FTO2B hepatoma cells through a mechanism involving ubiquitination and degradation of PTG. Furthermore, we demonstrate that the interaction between laforin and malin is a regulated process that is modulated by the AMP-activated protein kinase (AMPK). These findings provide further insights into the critical role of the laforin-malin complex in the control of glycogen metabolism and unravel a novel link between the energy sensor AMPK and glycogen metabolism. These data advance our understanding of the functional role of laforin and malin, which hopefully will facilitate the development of appropriate LD therapies.

  9. Remodeling of the Fission Yeast Cdc42 Cell-Polarity Module via the Sty1 p38 Stress-Activated Protein Kinase Pathway.

    PubMed

    Mutavchiev, Delyan R; Leda, Marcin; Sawin, Kenneth E

    2016-11-07

    The Rho family GTPase Cdc42 is a key regulator of eukaryotic cellular organization and cell polarity [1]. In the fission yeast Schizosaccharomyces pombe, active Cdc42 and associated effectors and regulators (the "Cdc42 polarity module") coordinate polarized growth at cell tips by controlling the actin cytoskeleton and exocytosis [2-4]. Localization of the Cdc42 polarity module to cell tips is thus critical for its function. Here we show that the fission yeast stress-activated protein kinase Sty1, a homolog of mammalian p38 MAP kinase, regulates localization of the Cdc42 polarity module. In wild-type cells, treatment with latrunculin A, a drug that leads to actin depolymerization, induces dispersal of the Cdc42 module from cell tips and cessation of polarized growth [5, 6]. We show that latrunculin A treatment also activates the Sty1 MAP kinase pathway and, strikingly, we find that loss of Sty1 MAP kinase signaling prevents latrunculin A-induced dispersal of the Cdc42 module, allowing polarized growth even in complete absence of the actin cytoskeleton. Regulation of the Cdc42 module by Sty1 is independent of Sty1's role in stress-induced gene expression. We also describe a system for activation of Sty1 kinase "on demand" in the absence of any external stress, and use this to show that Sty1 activation alone is sufficient to disperse the Cdc42 module from cell tips in otherwise unperturbed cells. During nitrogen-starvation-induced quiescence, inhibition of Sty1 converts non-growing, depolarized cells into growing, polarized cells. Our results place MAP kinase Sty1 as an important physiological regulator of the Cdc42 polarity module.

  10. Cytoplasmic Phospholipase A2 Modulation of Adolescent Rat Ethanol-Induced Protein Kinase C Translocation and Behavior

    PubMed Central

    Santerre, J. L.; Kolitz, E. B.; Pal, R.; Rogow, J. A.; Werner, D. F.

    2015-01-01

    Ethanol consumption typically begins during adolescence, a developmental period which exhibits many age-dependent differences in ethanol behavioral sensitivity. Protein kinase C (PKC) activity is largely implicated in ethanol-behaviors, and our previous work indicates that regulation of novel PKC isoforms likely contributes to decreased high-dose ethanol sensitivity during adolescence. The cytoplasmic Phospholipase A2 (cPLA2) signaling cascade selectivity modulates novel and atypical PKC isoform activity, as well as adolescent ethanol hypnotic sensitivity. Therefore, the current study was designed to ascertain adolescent cPLA2 activity both basally and in response to ethanol, as well as it's involvement in ethanol-induced PKC isoform translocation patterns. cPLA2 expression was elevated during adolescence, and activity was increased only in adolescents following high-dose ethanol administration. Novel, but not atypical PKC isoforms translocate to cytosolic regions following high-dose ethanol administration. Inhibiting cPLA2 with AACOCF3 blocked ethanol-induced PKC cytosolic translocation. Finally, inhibition of novel, but not atypical, PKC isoforms when cPLA2 activity was elevated, modulated adolescent high-dose ethanol-sensitivity. These data suggest that the cPLA2/PKC pathway contributes to the acute behavioral effects of ethanol during adolescence. PMID:25791059

  11. DNA Unwinding Functions of Minute Virus of Mice NS1 Protein Are Modulated Specifically by the Lambda Isoform of Protein Kinase C

    PubMed Central

    Dettwiler, Sabine; Rommelaere, Jean; Nüesch, Jürg P. F.

    1999-01-01

    The parvovirus minute virus of mice NS1 protein is a multifunctional protein involved in a variety of processes during virus propagation, ranging from viral DNA replication to promoter regulation and cytotoxic action to the host cell. Since NS1 becomes phosphorylated during infection, it was proposed that the different tasks of this protein might be regulated in a coordinated manner by phosphorylation. Indeed, comparing biochemical functions of native NS1 with its dephosphorylated counterpart showed that site-specific nicking of the origin and the helicase and ATPase activities are remarkably reduced upon NS1 dephosphorylation while site-specific affinity of the protein to the origin became enhanced. As a consequence, the dephosphorylated polypeptide is deficient for initiation of DNA replication. By adding fractionated cell extracts to a kinase-free in vitro replication system, the combination of two protein components containing members of the protein kinase C (PKC) family was found to rescue the replication activity of the dephosphorylated NS1 protein upon addition of PKC cofactors. One of these components, termed HA-1, also stimulated NS1 helicase function in response to acidic lipids but not phorbol esters, indicating the involvement of atypical PKC isoforms in the modulation of this NS1 function (J. P. F. Nüesch, S. Dettwiler, R. Corbau, and J. Rommelaere, J. Virol. 72:9966–9977, 1998). The present study led to the identification of atypical PKCλ/ι as the active component of HA-1 responsible for the regulation of NS1 DNA unwinding and replicative functions. Moreover, a target PKCλ phosphorylation site was localized at S473 of NS1. By site-directed mutagenesis, we showed that this residue is essential for NS1 helicase activity but not promoter regulation, suggesting a possible modulation of NS1 functions by PKCλ phosphorylation at residue S473. PMID:10438831

  12. Substance P modulates sensory action potentials in the lamprey via a protein kinase C-mediated reduction of a 4-aminopyridine-sensitive potassium conductance.

    PubMed

    Parker, D; Svensson, E; Grillner, S

    1997-10-01

    We have examined the effects of the tachykinin substance P on the action potential of lamprey mechanosensory dorsal cells. Substance P increased the spike duration and reduced the afterhyperpolarization. These effects were mimicked by stimulation of the dorsal root, which contains tachykinin-like immunoreactive fibres. The tachykinin antagonist spantide II blocked the effects of both substance P and dorsal root stimulation. The spike broadening was voltage-dependent, and was due to the reduction of a 4-aminopyridine-sensitive potassium conductance. The spike broadening was mimicked by G-protein activators and blocked by the G-protein inhibitor GDPbetaS. Pertussis toxin did not block the effects of substance P. The spike broadening was blocked by the protein kinase C and cAMP-dependent protein kinase inhibitor H7, and by the specific protein kinase C antagonist chelerythrine, but not by the cAMP and cGMP-dependent protein kinase inhibitor H8. The phorbol ester phorbol 12,13-dibutyrate mimicked and blocked the effects of substance P, supporting the role of protein kinase C in the spike modulation. The adenylate cyclase activator forskolin and the cAMP agonist SpcAMPs mimicked but did not block the effects of substance P on the spike duration, suggesting that protein kinase A also modulates the dorsal cell action potential, but that substance P acts independently of this pathway. Substance P also increased the excitability of the dorsal cells. This effect was blocked by 4-AP, PDBu and chelerythrine, but not by H8, suggesting that the increase in excitability shares the same intracellular and effector pathways as the spike broadening.

  13. Protein kinase A modulates transforming growth factor-β signaling through a direct interaction with Smad4 protein.

    PubMed

    Yang, Huibin; Li, Gangyong; Wu, Jing-Jiang; Wang, Lidong; Uhler, Michael; Simeone, Diane M

    2013-03-22

    Transforming growth factor β (TGFβ) signaling normally functions to regulate embryonic development and cellular homeostasis. It is increasingly recognized that TGFβ signaling is regulated by cross-talk with other signaling pathways. We previously reported that TGFβ activates protein kinase A (PKA) independent of cAMP through an interaction of an activated Smad3-Smad4 complex and the regulatory subunit of the PKA holoenzyme (PKA-R). Here we define the interaction domains of Smad4 and PKA-R and the functional consequences of this interaction. Using a series of Smad4 and PKA-R truncation mutants, we identified amino acids 290-300 of the Smad4 linker region as critical for the specific interaction of Smad4 and PKA-R. Co-immunoprecipitation assays showed that the B cAMP binding domain of PKA-R was sufficient for interaction with Smad4. Targeting of B domain regions conserved among all PKA-R isoforms and exposed on the molecular surface demonstrated that amino acids 281-285 and 320-329 were required for complex formation with Smad4. Interactions of these specific regions of Smad4 and PKA-R were necessary for TGFβ-mediated increases in PKA activity, CREB (cAMP-response element-binding protein) phosphorylation, induction of p21, and growth inhibition. Moreover, this Smad4-PKA interaction was required for TGFβ-induced epithelial mesenchymal transition, invasion of pancreatic tumor cells, and regulation of tumor growth in vivo.

  14. The synthetic genetic network around PKC1 identifies novel modulators and components of protein kinase C signaling in Saccharomyces cerevisiae.

    PubMed

    Krause, Sue A; Xu, Hong; Gray, Joseph V

    2008-11-01

    Budding yeast Saccharomyces cerevisiae contains one protein kinase C (PKC) isozyme encoded by the essential gene PKC1. Pkc1 is activated by the small GTPase Rho1 and plays a central role in the cell wall integrity (CWI) signaling pathway. This pathway acts primarily to remodel the cell surface throughout the normal life cycle and upon various environmental stresses. The pathway is heavily branched, with multiple nonessential branches feeding into and out of the central essential Rho1-Pkc1 module. In an attempt to identify novel components and modifiers of CWI signaling, we determined the synthetic lethal genetic network around PKC1 by using dominant-negative synthetic genetic array analysis. The resulting mutants are hypersensitive to lowered Pkc1 activity. The corresponding 21 nonessential genes are closely related to CWI function: 14 behave in a chemical-genetic epistasis test as acting in the pathway, and 6 of these genes encode known components. Twelve of the 21 null mutants display elevated CWI reporter activity, consistent with the idea that the pathway is activated by and compensates for loss of the gene products. Four of the 21 mutants display low CWI reporter activity, consistent with the idea that the pathway is compromised in these mutants. One of the latter group of mutants lacks Ack1(Ydl203c), an uncharacterized SEL-1 domain-containing protein that we find modulates pathway activity. Epistasis analysis places Ack1 upstream of Pkc1 in the CWI pathway and dependent on the upstream Rho1 GTP exchange factors Rom2 and Tus1. Overall, the synthetic genetic network around PKC1 directly and efficiently identifies known and novel components of PKC signaling in yeast.

  15. The p38 mitogen-activated protein kinase cascade modulates T helper type 17 differentiation and functionality in multiple sclerosis

    PubMed Central

    Di Mitri, Diletta; Sambucci, Manolo; Loiarro, Maria; De Bardi, Marco; Volpe, Elisabetta; Cencioni, Maria Teresa; Gasperini, Claudio; Centonze, Diego; Sette, Claudio; Akbar, Arne N; Borsellino, Giovanna; Battistini, Luca

    2015-01-01

    The p38 mitogen-activated protein kinase cascade is required for the induction of a T helper type 17 (Th17) -mediated autoimmune response, which underlies the development and progression of several autoimmune diseases, such as experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis (MS). However, the contribution of p38 phosphorylation to human Th cell differentiation has not been clarified. Here we demonstrate that the p38 signalling pathway is implicated in the generation of Th17 lymphocytes from human CD4+ CD27+ CD45RA+ naive T cells, both in healthy donors and in patients affected by the relapsing–remitting form of MS. Our data also indicate that p38 activation is essential for interleukin-17 release from central memory lymphocytes and committed Th17 cell clones. Furthermore, CD4+ T cells isolated from individuals with relapsing–remitting MS display an altered responsiveness of the p38 cascade, resulting in increased p38 phosphorylation upon stimulation. These findings suggest that the p38 signalling pathway, by modulating the Th17 differentiation and response, is involved in the pathogenesis of MS, and open new perspectives for the use of p38 inhibitors in the treatment of Th17-mediated autoimmune diseases. PMID:26095162

  16. Adenylate cyclase, cyclic AMP and extracellular-signal-regulated kinase-2 in airway smooth muscle: modulation by protein kinase C and growth serum.

    PubMed Central

    Moughal, N; Stevens, P A; Kong, D; Pyne, S; Pyne, N J

    1995-01-01

    Bradykinin and phorbol 12-myristate 13-acetate stimulate adenylate cyclase activity in serum-depleted cultured airway smooth muscle via a protein kinase C (PKC)-dependent pathway. The probable target is the type II adenylate cyclase, which can integrate coincident signals from both PKC and Gs. Therefore, activation of Gs (by cholera-toxin pre-treatment) amplified the bradykinin-stimulated cyclic AMP signal and concurrently attenuated the partial activation of extracellular-signal-regulated kinase-2 (ERK-2) by bradykinin. We have previously demonstrated that, in order to induce full activation of ERK-2 with bradykinin, it is necessary to obliterate PKC-stimulated cyclic AMP formation. We concluded that the cyclic AMP signal limits the magnitude of ERK-2 activation [Pyne, Moughal, Stevens, Tolan and Pyne (1994) Biochem. J. 304, 611-616]. The present study indicates that the bradykinin-stimulated ERK-2 pathway is entirely cyclic AMP-sensitive, and suggests that coincident signal detection by adenylate cyclase may be an important physiological route for the modulation of early mitogenic signalling. Furthermore, the direct inhibition of adenylate cyclase activity enables bradykinin to induce DNA synthesis, indicating that the PKC-dependent activation of adenylate cyclase limits entry of cells into the cell cycle. These studies suggest that the mitogenicity of an agonist may be governed, in part, by its ability to stimulate an inhibitory cyclic AMP signal pathway in the cell. The activation of adenylate cyclase by PKC appears to be downstream of phospholipase D. However, in cells that were maintained in growth serum (i.e. were not growth-arrested), bradykinin was unable to elicit a PKC-stimulated cyclic AMP response. The lesion in the signal-response coupling was not at the level of either the receptor or phospholipase D, which remain functionally operative and suggests modification occurs at either PKC or adenylate cyclase itself. These studies are discussed with

  17. Protein kinase CK2 modulates IL-6 expression in inflammatory breast cancer

    SciTech Connect

    Drygin, Denis Ho, Caroline B.; Omori, Mayuko; Bliesath, Joshua; Proffitt, Chris; Rice, Rachel; Siddiqui-Jain, Adam; O'Brien, Sean; Padgett, Claire; Lim, John K.C.; Anderes, Kenna; Rice, William G.; Ryckman, David

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer We examine the potential cross-talk between CK2 and IL-6. Black-Right-Pointing-Pointer Inhibition of CK2 by siRNA or CX-4945 inhibits expression of IL-6 in models of IBC. Black-Right-Pointing-Pointer Treatment of IBC patient in the clinic with CX-4945 reduces her IL-6 plasma levels. Black-Right-Pointing-Pointer We demonstrate that CK2 is a potential therapeutic target for IL-6 driven diseases. -- Abstract: Inflammatory breast cancer is driven by pro-angiogenic and pro-inflammatory cytokines. One of them Interleukin-6 (IL-6) is implicated in cancer cell proliferation and survival, and promotes angiogenesis, inflammation and metastasis. While IL-6 has been shown to be upregulated by several oncogenes, the mechanism behind this phenomenon is not well characterized. Here we demonstrate that the pleotropic Serine/Threonine kinase CK2 is implicated in the regulation of IL-6 expression in a model of inflammatory breast cancer. We used siRNAs targeted toward CK2 and a selective small molecule inhibitor of CK2, CX-4945, to inhibit the expression and thus suppress the secretion of IL-6 in in vitro as well as in vivo models. Moreover, we report that in a clinical trial, CX-4945 was able to dramatically reduce IL-6 levels in plasma of an inflammatory breast cancer patient. Our data shed a new light on the regulation of IL-6 expression and position CX-4945 and potentially other inhibitors of CK2, for the treatment of IL-6-driven cancers and possibly other diseases where IL-6 is instrumental, including rheumatoid arthritis.

  18. Protein kinase biochemistry and drug discovery.

    PubMed

    Schwartz, Phillip A; Murray, Brion W

    2011-12-01

    Protein kinases are fascinating biological catalysts with a rapidly expanding knowledge base, a growing appreciation in cell regulatory control, and an ascendant role in successful therapeutic intervention. To better understand protein kinases, the molecular underpinnings of phosphoryl group transfer, protein phosphorylation, and inhibitor interactions are examined. This analysis begins with a survey of phosphate group and phosphoprotein properties which provide context to the evolutionary selection of phosphorylation as a central mechanism for biological regulation of most cellular processes. Next, the kinetic and catalytic mechanisms of protein kinases are examined with respect to model aqueous systems to define the elements of catalysis. A brief structural biology overview further delves into the molecular basis of catalysis and regulation of catalytic activity. Concomitant with a prominent role in normal physiology, protein kinases have important roles in the disease state. To facilitate effective kinase drug discovery, classic and emerging approaches for characterizing kinase inhibitors are evaluated including biochemical assay design, inhibitor mechanism of action analysis, and proper kinetic treatment of irreversible inhibitors. As the resulting protein kinase inhibitors can modulate intended and unintended targets, profiling methods are discussed which can illuminate a more complete range of an inhibitor's biological activities to enable more meaningful cellular studies and more effective clinical studies. Taken as a whole, a wealth of protein kinase biochemistry knowledge is available, yet it is clear that a substantial extent of our understanding in this field remains to be discovered which should yield many new opportunities for therapeutic intervention.

  19. The phosphotransferase protein EIIA(Ntr) modulates the phosphate starvation response through interaction with histidine kinase PhoR in Escherichia coli.

    PubMed

    Lüttmann, Denise; Göpel, Yvonne; Görke, Boris

    2012-10-01

    Many Proteobacteria possess the paralogous PTS(Ntr), in addition to the sugar transport phosphotransferase system (PTS). In the PTS(Ntr) phosphoryl-groups are transferred from phosphoenolpyruvate to protein EIIA(Ntr) via the phosphotransferases EI(Ntr) and NPr. The PTS(Ntr) has been implicated in regulation of diverse physiological processes. In Escherichia coli, the PTS(Ntr) plays a role in potassium homeostasis. In particular, EIIA(Ntr) binds to and stimulates activity of a two-component histidine kinase (KdpD) resulting in increased expression of the genes encoding the high-affinity K(+) transporter KdpFABC. Here, we show that the phosphate (pho) regulon is likewise modulated by PTS(Ntr). The pho regulon, which comprises more than 30 genes, is activated by the two-component system PhoR/PhoB under conditions of phosphate starvation. Mutants lacking EIIA(Ntr) are unable to fully activate the pho genes and exhibit a growth delay upon adaptation to phosphate limitation. In contrast, pho expression is increased above the wild-type level in mutants deficient for EIIA(Ntr) phosphorylation suggesting that non-phosphorylated EIIA(Ntr) modulates pho. Protein interaction analyses reveal binding of EIIA(Ntr) to histidine kinase PhoR. This interaction increases the amount of phosphorylated response regulator PhoB. Thus, EIIA(Ntr) is an accessory protein that modulates the activities of two distinct sensor kinases, KdpD and PhoR, in E. coli.

  20. Identification of Novel Coxiella burnetii Icm/Dot Effectors and Genetic Analysis of Their Involvement in Modulating a Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Lifshitz, Ziv; Burstein, David; Schwartz, Kierstyn; Shuman, Howard A.; Pupko, Tal

    2014-01-01

    Coxiella burnetii, the causative agent of Q fever, is a human intracellular pathogen that utilizes the Icm/Dot type IVB secretion system to translocate effector proteins into host cells. To identify novel C. burnetii effectors, we applied a machine-learning approach to predict C. burnetii effectors, and examination of 20 such proteins resulted in the identification of 13 novel effectors. To determine whether these effectors, as well as several previously identified effectors, modulate conserved eukaryotic pathways, they were expressed in Saccharomyces cerevisiae. The effects on yeast growth were examined under regular growth conditions and in the presence of caffeine, a known modulator of the yeast cell wall integrity (CWI) mitogen-activated protein (MAP) kinase pathway. In the presence of caffeine, expression of the effectors CBU0885 and CBU1676 caused an enhanced inhibition of yeast growth, and the growth inhibition of CBU0388 was suppressed. Furthermore, analysis of synthetic lethality effects and examination of the activity of the CWI MAP kinase transcription factor Rlm1 indicated that CBU0388 enhances the activation of this MAP kinase pathway in yeast, while CBU0885 and CBU1676 abolish this activation. Additionally, coexpression of CBU1676 and CBU0388 resulted in mutual suppression of their inhibition of yeast growth. These results strongly indicate that these three effectors modulate the CWI MAP kinase pathway in yeast. Moreover, both CBU1676 and CBU0885 were found to contain a conserved haloacid dehalogenase (HAD) domain, which was found to be required for their activity. Collectively, our results demonstrate that MAP kinase pathways are most likely targeted by C. burnetii Icm/Dot effectors. PMID:24958706

  1. Sensitization of human colon cancer cells to sodium butyrate-induced apoptosis by modulation of sphingosine kinase 2 and protein kinase D

    SciTech Connect

    Xiao, Min; Liu, Yungang; Zou, Fei

    2012-01-01

    Sphingosine kinases (SphKs) have been recognized as important proteins regulating cell proliferation and apoptosis. Of the two isoforms of SphK (SphK1 and SphK2), little is known about the functions of SphK2. Sodium butyrate (NaBT) has been established as a promising chemotherapeutic agent, but the precise mechanism for its effects is unknown. In this study, we investigated the role of SphK2 in NaBT-induced apoptosis of HCT116 colon cancer cells. The results indicated that following NaBT treatment SphK2 was translocated from the nucleus to the cytoplasm, leading to its accumulation in the cytoplasm; in the meantime, only mild apoptosis occurred. However, downregulation of SphK2 resulted in sensitized apoptosis, and overexpression of SphK2 led to even lighter apoptosis; these strongly indicate an inhibitory role of SphK2 in cell apoptosis induced by NaBT. After knocking down protein kinase D (PKD), another protein reported to be critical in cell proliferation/apoptosis process, by using siRNA, blockage of cytoplasmic accumulation of SphK2 and sensitized apoptosis following NaBT treatment were observed. The present study suggests that PKD and SphK2 may form a mechanism for the resistance of cancer cells to tumor chemotherapies, such as HCT116 colon cancer cells to NaBT, and these two proteins may become molecular targets for designation of new tumor-therapeutic drugs. -- Highlights: Black-Right-Pointing-Pointer In the present study sodium butyrate (10 mM) induced mild apoptosis of cancer cells. Black-Right-Pointing-Pointer The apoptosis was negatively regulated by cytoplasmic Sphingosine Kinase 2 (SphK2). Black-Right-Pointing-Pointer Translocation of SphK2 from nucleus to cytoplasm was mediated by protein kinase D. Black-Right-Pointing-Pointer Downregulation of SphK2 or protein kinase D leads to sensitized cell apoptosis.

  2. SDF-1-induced adhesion of monocytes to vascular endothelium is modulated by azelnidipine via protein kinase C inhibition.

    PubMed

    Takahashi, Keiko; Shimokado, Kentaro; Yoshida, Masayuki

    2006-12-15

    Monocyte-endothelial interaction and its modulation by chemokines play a key role in atherogenesis and inflammation. We examined the potential effects of stromal cell-derived factor (SDF-1) and azelnidipine, a novel dihydropyridine derivative, toward monocyte-endothelial interaction. Human monocytes were prepared from peripheral blood mononuclear cells obtained from healthy volunteers and pretreated with azelnidipine (1 microM) for 48 h, after which their adhesion to interleukin-1beta (IL-1beta)-activated human umbilical vein endothelial cells (HUVECs) was analyzed using an in vitro flow apparatus with a shear stress of 1 dyn/cm(2). In some experiments, monocytes were incubated in the presence of stromal cell-derived factor (SDF-1), a chemokine, just prior to the assay. Pre-incubation of monocytes with SDF-1 enhanced their adhesion to activated HUVECs. When monocytes were pre-incubated in the presence of azelnidipine, baseline levels as well as SDF-1-induced monocyte adhesion levels were reduced. Interestingly, the surface expressions of the adhesion molecules CD11a, CD11b, and CD36, were not changed by azelnidipine treatment. Western blotting analysis revealed that activation of protein kinase C (PKC)alpha was inhibited by azelnidipine treatment, while it also reduced the SDF-1-induced increase in intracellular calcium concentration ([Ca(2+)](i)). Further, pre-incubation of monocytes with Go6976, a potent inhibitor of PKCalpha, significantly reduced monocyte adhesion to HUVECs. Our results demonstrated an inhibitory action of azelnidipine toward adhesive interactions of monocytes to HUVECs, which involves inhibition of PKCalpha and a reduction in [Ca(2+)](i). These findings imply a protective role of azelnidipine against inflammation in atherosclerosis.

  3. Probing the mechanisms underlying modulation of quinidine sensitivity to cardiac IKs block by protein kinase A-mediated IKs phosphorylation

    PubMed Central

    Yang, Tao; Kanki, Hideaki; Zhang, Wei; Roden, Dan M

    2009-01-01

    Background and purpose: Cardiac IKs is enhanced by protein kinase A (PKA) stimulation. And PKA-stimulated IKs is about threefold less sensitive to quinidine block than basal current. In this study, we further tested two competing hypotheses: IKs phosphorylation either (i) modulates access of blocking drugs to a binding site; or (ii) destabilizes the drug–channel interaction. Experimental approach: To distinguish between these hypotheses, we studied quinidine block of IKs channels in which three PKA site residues of the α-subunit KCNQ1 were mutated with a bulky negative charged aspartic acid (D). To study alleviation of IKs block by quinidine, we compared activating current at +60 mV, either with or without 5 s hyperpolarizing prepulses to −120 mV. Key results: Without PKA stimulation, quinidine (100 µM) blocked wild-type current to a similar extent with and without the prepulse (93 ± 2% of pre-drug current at +60 mV vs. 95 ± 1%). With PKA-stimulated wild-type channels, however, there was less block with the hyperpolarization to −120 mV: at +60 mV, block was 71 ± 2% (−prepulse) versus 58 ± 3% (+prepulse). Individual D-mutations and the triple-D mutant were resistant to quinidine block similar to that seen with PKA-stimulated wild-type IKs. Conclusions and implications: We conclude that phosphorylation-induced insertion of bulky negative charges alleviates quinidine block and that PKA-induced stimulation, by conferring negative charges to the channels, blunts IKs block as the interaction between the channels and blockers becomes destabilized. These effects would be of clinical significance in providing protective mechanisms against pro-arrhythmias caused by drug-induced inhibition of IKs and IKr. PMID:19522859

  4. Modulation of histamine-induced Ca2+ release by protein kinase C. Effects on cytosolic and mitochondrial [Ca2+] peaks.

    PubMed

    Montero, Mayte; Lobatón, Carmen D; Gutierrez-Fernández, Silvia; Moreno, Alfredo; Alvarez, Javier

    2003-12-12

    In HeLa cells, histamine induces production of inositol 1,4,5-trisphosphate (InsP3) and release of Ca2+ from the endoplasmic reticulum (ER). Ca2+ release is typically biphasic, with a fast and brief initial phase, followed by a much slower and prolonged one. In the presence of inhibitors of protein kinase C (PKC), including staurosporine and the specific inhibitors GF109203X and Ro-31-8220, the fast phase continued until the ER became fully empty. On the contrary, treatment with phorbol 12,13-dibutyrate inhibited Ca2+ release. Staurosporine had no effect on InsP3-induced Ca2+ release in permeabilized cells and did not modify either histamine-induced InsP3 production. These data suggest that histamine induces Ca2+ release and with a short lag activates PKC to down-regulate it. Consistently, Ca2+ oscillations induced by histamine were increased in amplitude and decreased in frequency in the presence of PKC inhibitors. We show also that mitochondrial [Ca2+] was much more sensitive to changes in ER-Ca2+ release induced by PKC modulation than cytosolic [Ca2+]. PKC inhibitors increased the histamine-induced mitochondrial [Ca2+] peak by 4-fold but increased the cytosolic [Ca2+] peak only by 20%. On the contrary, PKC activation inhibited the mitochondrial [Ca2+] peak by 90% and the cytosolic one by only 50%. Similarly, the combination of PKC inhibitors with the mitochondrial Ca2+ uniporter activator SB202190 led to dramatic increases in mitochondrial [Ca2+] peaks, with little effect on cytosolic ones. This suggests that activation of ER-Ca2+ release by PKC inhibitors could be involved in apoptosis induced by staurosporine. In addition, these mechanisms allow flexible and independent regulation of cytosolic and mitochondrial [Ca2+] during cell stimulation.

  5. Imposed glutathione-mediated redox switch modulates the tobacco wound-induced protein kinase and salicylic acid-induced protein kinase activation state and impacts on defence against Pseudomonas syringae.

    PubMed

    Matern, Sanja; Peskan-Berghoefer, Tatjana; Gromes, Roland; Kiesel, Rebecca Vazquez; Rausch, Thomas

    2015-04-01

    The role of the redox-active tripeptide glutathione in plant defence against pathogens has been studied extensively; however, the impact of changes in cellular glutathione redox potential on signalling processes during defence reactions has remained elusive. This study explored the impact of elevated glutathione content on the cytosolic redox potential and on early defence signalling at the level of mitogen-activated protein kinases (MAPKs), as well as on subsequent defence reactions, including changes in salicylic acid (SA) content, pathogenesis-related gene expression, callose depositions, and the hypersensitive response. Wild-type (WT) Nicotiana tabacum L. and transgenic high-glutathione lines (HGL) were transformed with the cytosol-targeted sensor GRX1-roGFP2 to monitor the cytosolic redox state. Surprisingly, HGLs displayed an oxidative shift in their cytosolic redox potential and an activation of the tobacco MAPKs wound-induced protein kinase (WIPK) and SA-induced protein kinase (SIPK). This activation occurred in the absence of any change in free SA content, but was accompanied by constitutively increased expression of several defence genes. Similarly, rapid activation of MAPKs could be induced in WT tobacco by exposure to either reduced or oxidized glutathione. When HGL plants were challenged with adapted or non-adapted Pseudomonas syringae pathovars, the cytosolic redox shift was further amplified and the defence response was markedly increased, showing a priming effect for SA and callose; however, the initial and transient hyperactivation of MAPK signalling was attenuated in HGLs. The results suggest that, in tobacco, MAPK and SA signalling may operate independently, both possibly being modulated by the glutathione redox potential. Possible mechanisms for redox-mediated MAPK activation are discussed.

  6. Nintedanib modulates surfactant protein-D expression in A549 human lung epithelial cells via the c-Jun N-terminal kinase-activator protein-1 pathway.

    PubMed

    Kamio, Koichiro; Usuki, Jiro; Azuma, Arata; Matsuda, Kuniko; Ishii, Takeo; Inomata, Minoru; Hayashi, Hiroki; Kokuho, Nariaki; Fujita, Kazue; Saito, Yoshinobu; Miya, Toshimichi; Gemma, Akihiko

    2015-06-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a high mortality rate. Signalling pathways activated by several tyrosine kinase receptors are known to be involved in lung fibrosis, and this knowledge has led to the development of the triple tyrosine kinase inhibitor nintedanib, an inhibitor of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), for the treatment of IPF. Pulmonary surfactant protein D (SP-D), an important biomarker of IPF, reportedly attenuates bleomycin-induced pulmonary fibrosis in mice. In this study, we investigated whether nintedanib modulates SP-D expression in human lung epithelial (A549) cells using quantitative real-time reverse transcriptase polymerase chain reaction and western blotting. To investigate the mechanisms underlying the effects of nintedanib, we evaluated the phosphorylation of c-Jun N-terminal kinase (JNK) and its downstream target c-Jun. The effect of the JNK inhibitor SP600125 on c-Jun phosphorylation was also tested. Activation of activator protein-1 (AP-1) was examined using an enzyme-linked immunosorbent assay-based test, and cell proliferation assays were performed to estimate the effect of nintedanib on cell proliferation. Furthermore, we treated mice with nintedanib to examine its in vivo effect on SP-D levels in lungs. These experiments showed that nintedanib up-regulated SP-D messenger RNA expression in a dose-dependent manner at concentrations up to 5 μM, with significant SP-D induction observed at concentrations of 3 μM and 5 μM, in comparison with that observed in vehicle controls. Nintedanib stimulated a rapid increase in phosphorylated JNK in A549 cells within 30 min of treatment and stimulated c-Jun phosphorylation, which was inhibited by the JNK inhibitor SP600125. Additionally, nintedanib was found to activate AP-1. A549 cell proliferation was not affected by nintedanib at any of the tested

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

    PubMed Central

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

    2016-01-01

    The heterotrimeric AMP-activated protein kinase (AMPK), consisting of α, β and γ subunits, is a stress-sensing enzyme that is activated by phosphorylation of its activation loop in response to increases in cellular AMP. N-terminal myristoylation of the β-subunit has been shown to suppress Thr172 phosphorylation, keeping AMPK in an inactive state. Here we use amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the structural and dynamic properties of the mammalian myristoylated and non-myristoylated inactivated AMPK (D139A) in the presence and absence of nucleotides. HDX MS data suggests that the myristoyl group binds near the first helix of the C-terminal lobe of the kinase domain similar to other kinases. Our data, however, also shows that ATP.Mg2+ results in a global stabilization of myristoylated, but not non-myristoylated AMPK, and most notably for peptides of the activation loop of the α-kinase domain, the autoinhibitory sequence (AIS) and the βCBM. AMP does not have that effect and HDX measurements for myristoylated and non-myristoylated AMPK in the presence of AMP are similar. These differences in dynamics may account for a reduced basal rate of phosphorylation of Thr172 in myristoylated AMPK in skeletal muscle where endogenous ATP concentrations are very high. PMID:28000716

  8. Protein kinase C epsilon is localized to the Golgi via its zinc-finger domain and modulates Golgi function.

    PubMed Central

    Lehel, C; Olah, Z; Jakab, G; Anderson, W B

    1995-01-01

    Protein kinase C (PKC) is a multigene family of serine/threonine kinases that are central to many signal transduction pathways. Among the PKC isozymes, only PKC epsilon has been reported to exhibit full oncogenic potential. PKC epsilon also displays unique substrate specificity and intracellular localization. To examine the interrelationship between the biological effects and domain structure of PKC epsilon, NIH 3T3 cells were stably transfected to overexpress different epitope-tagged fragments of PKC epsilon. The overexpressed proteins each contain the epsilon-tag peptide at the C terminus to allow ready detection with an antibody specific for the tag. The holo-PKC epsilon was found to localize with the Golgi network and other compartments, whereas the zinc-finger domain localized exclusively at the Golgi. Golgi-specific glycosaminoglycan sulfation was strongly inhibited in cells overexpressing either holo-PKC epsilon or its zinc-finger domain, while the secretion of sulfated glycosaminoglycans into the medium was impaired in cells expressing the PKC epsilon zinc-finger domain. Thus, these results suggest that PKC epsilon may be involved in specifically regulating Golgi-related processes. Further, the results indicate that PKC epsilon domains other than the kinase domain may also have biological activity and that the zinc-finger domain may function as a subcellular localization signal. Images Fig. 1 Fig. 2 Fig. 3 PMID:7877991

  9. Protein kinase C modulates Aurora-kinase inhibition induced by CCT129202 in HMC-1⁵⁶⁰,⁸¹⁶ cell line.

    PubMed

    Tobío, Araceli; Alfonso, Amparo; Fernández-Araujo, Andrea; Alonso, Eva; Botana, Luis M

    2013-01-01

    The human mast cell line HMC-1⁵⁶⁰,⁸¹⁶ carries activating mutations in the proto-oncogene of c-kit that cause autophosphorylation and permanent c-kit receptor activation. The compound CCT129202 is a new and selective inhibitor of Aurora kinase A and B that decreases the viability of a variety of human tumor cell lines. The effect of Aurora kinase inhibition was assessed in the HMC-1⁵⁶⁰,⁸¹⁶ line in order to find a suitable tool for mastocytosis treatment. CCT129202 treatment induces a significant decrease in cell viability in HMC-1⁵⁶⁰,⁸¹⁶ cells after 48 hours of treatment. Moreover, caspase-3 and caspase-8 activation was induced after incubation of HMC-1⁵⁶⁰,⁸¹⁶ cells in the presence of CCT129202. It has been demonstrated that Protein Kinase C (PKC) plays a crucial role in mast cell activation as well as cell migration, adhesion and apoptotic cell death. Co-treatment of Ca²⁺-independent PKCs (δ ε and θ) inhibitor GF109203X with CCT129202, reduces caspase-3 activation which controls cell levels. In contrast, Go6976, an inhibitor of Ca²⁺-dependent PKCs, increases caspase-3 activation. Oppositely, GF109203X does not modify CCT129202-induced apoptosis through the caspase-8 pathway whereas Go6976 treatment abolishes the increase on caspase-8 activity due to CCT129202. This implies that Ca²⁺-independent PKC isoforms seems to be related with CCT129202-induced apoptosis through the caspase- 3 pathway, whereas Ca²⁺-dependent PKC isoforms are related with the CCT129202 effect on the caspase-8 pathway. Interestingly, CCT129202 cytotoxic effect remains even though Ca²⁺-dependent PKCs are inhibited, which shows that the Aurora kinase inhibitor effect is acting through the caspase-3 pathway. On the other hand, Ca²⁺-independent PKCs inhibition does not affect the final apoptotic CCT129202 effect because this seems to be mediated by the caspase-8 pathway. Moreover, CCT129202 does not affect PKCδ and Ca

  10. MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase.

    PubMed Central

    Stokoe, D; Campbell, D G; Nakielny, S; Hidaka, H; Leevers, S J; Marshall, C; Cohen, P

    1992-01-01

    A novel protein kinase, which was only active when phosphorylated by the mitogen-activated protein kinase (MAP kinase), has been purified 85,000-fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase-2, was distinguished from S6 kinase-II (MAPKAP kinase-1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase-2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase-2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase-2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase-2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor-stimulated protein kinase cascade bifurcates. Images PMID:1327754

  11. Rac2 GTPase activation by angiotensin II is modulated by Ca2+/calcineurin and mitogen-activated protein kinases in human neutrophils.

    PubMed

    El Bekay, Rajaa; Alba, Gonzalo; Reyes, M Edith; Chacón, Pedro; Vega, Antonio; Martín-Nieto, José; Jiménez, Juan; Ramos, Eladio; Oliván, Josefina; Pintado, Elízabeth; Sobrino, Francisco

    2007-11-01

    Angiotensin II (Ang II) highly stimulates superoxide anion production by neutrophils. The G-protein Rac2 modulates the activity of NADPH oxidase in response to various stimuli. Here, we describe that Ang II induced both Rac2 translocation from the cytosol to the plasma membrane and Rac2 GTP-binding activity. Furthermore, Clostridium difficile toxin A, an inhibitor of the Rho-GTPases family Rho, Rac and Cdc42, prevented Ang II-elicited O2-/ROS production, phosphorylation of the mitogen-activated protein kinases (MAPKs) p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2, and Rac2 activation. Rac2 GTPase inhibition by C. difficile toxin A was accompanied by a robust reduction of the cytosolic Ca(2)(+) elevation induced by Ang II in human neutrophils. Furthermore, SB203580 and PD098059 act as inhibitors of p38MAPK and ERK1/2 respectively, wortmannin, an inhibitor of phosphatidylinositol-3-kinase, and cyclosporin A, a calcineurin inhibitor, hindered both translocation of Rac2 from the cytosol to the plasma membrane and enhancement of Rac2 GTP-binding elicited by Ang II. These results provide evidence that the activation of Rac2 by Ang II is exerted through multiple signalling pathways, involving Ca(2)(+)/calcineurin and protein kinases, the elucidation of which should be insightful in the design of new therapies aimed at reversing the inflammation of vessel walls found in a number of cardiovascular diseases.

  12. Hexamethylenebisacetamide modulation of thyroglobulin and protein levels in thyroid cells is not mediated by phosphatidylinositol-3-kinase: a study with wortmannin.

    PubMed

    Aouani, A; Samih, N; Amphoux-Fazekas, T; Hovsépian, S; Fayet, G

    1999-04-01

    Hexamethylenebisacetamide (HMBA) induces in murine erythroleukemia cells (MELC) the commitment to terminal differentiation leading to globin gene expression. In the thyroid, HMBA acts as a growth factor and also as a differentiating agent. In the present paper, we studied the effect of HMBA on the very specific thyroid marker thyroglobulin (Tg) in two different thyroid cell systems, i.e., porcine cells in primary culture and ovine cells in long term culture. Using wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, we investigated whether this enzyme is involved in HMBA mode of action. We found that HMBA is a positive modulator of Tg production in porcine cells, but a negative effector in the OVNIS cell line. As all HMBA effects studied in the present paper, i.e., Tg production and total protein levels, are not inhibited by wortmannin, we suggest the non-involvement of phosphatidylinositol-3-kinase in HMBA mode of action.

  13. Extracellular Signal-regulated Kinases (ERKs) Phosphorylate Lin28a Protein to Modulate P19 Cell Proliferation and Differentiation.

    PubMed

    Liu, Xiangyuan; Chen, Min; Li, Long; Gong, Liyan; Zhou, Hu; Gao, Daming

    2017-03-10

    Lin28a, originally discovered in the nematode Caenorhabditis elegans and highly conserved across species, is a well characterized regulator of let-7 microRNA (miRNA) and is implicated in cell proliferation and pluripotency control. However, little is known about how Lin28a function is modulated at the post-translational level and thereby responds to major signaling pathways. Here we show that Lin28a is directly phosphorylated by ERK1/2 kinases at Ser-200. By editing lin28a gene with the CRISPR/Cas9-based method, we generated P19 mouse embryonic carcinoma stem cells expressing Lin28a-S200A (phospho-deficient) and Lin28a-S200D (phospho-mimetic) mutants, respectively, to study the functional impact of Ser-200 phosphorylation. Lin28a-S200D-expressing cells, but not Lin28a-S200A-expressing or control P19 embryonic carcinoma cells, displayed impaired inhibition of let-7 miRNA and resulted in decreased cyclin D1, whereas Lin28a-S200A knock-in cells expressed less let-7 miRNA, proliferated faster, and exhibited differentiation defect upon retinoic acid induction. Therefore our results support that ERK kinase-mediated Lin28a phosphorylation may be an important mechanism for pluripotent cells to facilitate the escape from the self-renewal cycle and start the differentiation process.

  14. Neuronal migration and protein kinases

    PubMed Central

    Ohshima, Toshio

    2015-01-01

    The formation of the six-layered structure of the mammalian cortex via the inside-out pattern of neuronal migration is fundamental to neocortical functions. Extracellular cues such as Reelin induce intracellular signaling cascades through the protein phosphorylation. Migrating neurons also have intrinsic machineries to regulate cytoskeletal proteins and adhesion properties. Protein phosphorylation regulates these processes. Moreover, the balance between phosphorylation and dephosphorylation is modified by extracellular cues. Multipolar-bipolar transition, radial glia-guided locomotion and terminal translocation are critical steps of radial migration of cortical pyramidal neurons. Protein kinases such as Cyclin-dependent kinase 5 (Cdk5) and c-Jun N-terminal kinases (JNKs) involve these steps. In this review, I shall give an overview the roles of protein kinases in neuronal migration. PMID:25628530

  15. Modulation of Purinergic Neuromuscular Transmission by Phorbol Dibutyrate is Independent of Protein Kinase C in Murine Urinary Bladder

    PubMed Central

    Silinsky, E. M.

    2012-01-01

    Parasympathetic control of murine urinary bladder consists of contractile components mediated by both muscarinic and purinergic receptors. Using intracellular recording techniques, the purinergic component of transmission was measured as both evoked excitatory junctional potentials (EJPs) in response to electrical field stimulation and spontaneous events [spontaneous EJPs (sEJPs)]. EJPs, but not sEJPs, were abolished by the application of the Na+ channel blocker tetrodotoxin and the Ca2+ channel blocker Cd2+. Both EJPs and sEJPs were abolished by the application of the P2X1 antagonist 8,8′-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt (NF279). Application of phorbol dibutyrate (PDBu) increased electrically evoked EJP amplitudes with no effect on mean sEJP amplitudes. Similar increases in EJP amplitudes were produced by PDBu in the presence of either the nonselective protein kinase inhibitor staurosporine or the specific protein kinase C (PKC) inhibitor 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X). These results suggest that PDBu increases the purinergic component of detrusor transmission through increasing neurogenic ATP release via a PKC-independent mechanism. PMID:22547572

  16. Extract of Polygala tenuifolia Alleviates Stress-Exacerbated Atopy-Like Skin Dermatitis through the Modulation of Protein Kinase A and p38 Mitogen-Activated Protein Kinase Signaling Pathway

    PubMed Central

    Sur, Bongjun; Lee, Bombi; Yoon, Ye Seul; Lim, Pooreum; Hong, Riwon; Yeom, Mijung; Lee, Hyang Sook; Park, Hijoon; Shim, Insop; Lee, Hyejung; Jang, Young Pyo; Hahm, Dae-Hyun

    2017-01-01

    Atopic dermatitis (AD) and stress create a vicious cycle: stress exacerbates atopic symptoms, and atopic disease elicits stress and anxiety. Targeting multiple pathways including stress and allergic inflammation is, therefore, important for treating AD. In this study, we investigated the remedial value of Polygala tenuifolia Willd. (PTW) for treating immobilization (IMO) stress-exacerbated atopy-like skin dermatitis and its underlying mechanism. Trimellitic anhydride (TMA) was applied to dorsal skin for sensitization and subsequently both ears for eliciting T-cell-dependent contact hypersensitivity in mice, which underwent 2 h-IMO stress and PTW administration for the latter 6 and 9 days in the ear exposure period of TMA, respectively. To elicit in vitro degranulation of human mast cell line-1 (HMC-1), 10 µM substance P (SP) and 200 nM corticotrophin-releasing factor (CRF) were sequentially added with 48 h-interval. PTW extract (500 µg/mL) was added 30 min before CRF treatment. IMO stress exacerbated TMA-induced scratching behavior by 252%, and increased their blood corticosterone levels by two-fold. Treatment with 250 mg/kg PTW significantly restored IMO stress-exacerbated scratching behavior and other indicators such as skin inflammation and water content, lymph node weights, and serum histamine and immunoglobulin E (lgE) levels. Furthermore, it also reversed TMA-stimulated expression of tumor necrosis factor (TNF)-α and interleukin (IL)-4 mRNAs in ear tissues. PTW significantly inhibited SP/CRF-stimulated degranulation of HMC-1 cells, subsequent tryptase secretion, and protein kinase A (PKA) activity. PTW also selectively inhibited p38 mitogen-activated protein kinase (MAPK) phosphorylation in SP/CRF-treated HMC-1 cells. PTW significantly inhibited HMC-1 cell degranulation and alleviated IMO stress-exacerbated atopic dermatitis symptoms by modulating the PKA/p38 MAPK signaling pathway. PMID:28106783

  17. Chickpea transcription factor CaTLP1 interacts with protein kinases, modulates ROS accumulation and promotes ABA-mediated stomatal closure

    PubMed Central

    Wardhan, Vijay; Pandey, Aarti; Chakraborty, Subhra; Chakraborty, Niranjan

    2016-01-01

    Tubby and Tubby-like proteins (TLPs), in mammals, play critical roles in neural development, while its function in plants is largely unknown. We previously demonstrated that the chickpea TLP, CaTLP1, participates in osmotic stress response and might be associated with ABA-dependent network. However, how CaTLP1 is connected to ABA signaling remains unclear. The CaTLP1 was found to be engaged in ABA-mediated gene expression and stomatal closure. Complementation of the yeast yap1 mutant with CaTLP1 revealed its role in ROS scavenging. Furthermore, complementation of Arabidopsis attlp2 mutant displayed enhanced stress tolerance, indicating the functional conservation of TLPs across the species. The presence of ABA-responsive element along with other motifs in the proximal promoter regions of TLPs firmly established their involvement in stress signalling pathways. The CaTLP1 promoter driven GUS expression was restricted to the vegetative organs, especially stem and rosette leaves. Global protein expression profiling of wild-type, attlp2 and complemented Arabidopsis plants revealed 95 differentially expressed proteins, presumably involved in maintaining physiological and biological processes under dehydration. Immunoprecipitation assay revealed that protein kinases are most likely to interact with CaTLP1. This study provides the first demonstration that the TLPs act as module for ABA-mediated stomatal closure possibly via interaction with protein kinase. PMID:27934866

  18. Non-degradative Ubiquitination of Protein Kinases

    PubMed Central

    Ball, K. Aurelia; Johnson, Jeffrey R.; Lewinski, Mary K.; Guatelli, John; Verschueren, Erik; Krogan, Nevan J.; Jacobson, Matthew P.

    2016-01-01

    Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well. PMID:27253329

  19. A Novel Mechanism of Ion Homeostasis and Salt Tolerance in Yeast: the Hal4 and Hal5 Protein Kinases Modulate the Trk1-Trk2 Potassium Transporter

    PubMed Central

    Mulet, Jose M.; Leube, Martin P.; Kron, Stephen J.; Rios, Gabino; Fink, Gerald R.; Serrano, Ramon

    1999-01-01

    The regulation of intracellular ion concentrations is a fundamental property of living cells. Although many ion transporters have been identified, the systems that modulate their activity remain largely unknown. We have characterized two partially redundant genes from Saccharomyces cerevisiae, HAL4/SAT4 and HAL5, that encode homologous protein kinases implicated in the regulation of cation uptake. Overexpression of these genes increases the tolerance of yeast cells to sodium and lithium, whereas gene disruptions result in greater cation sensitivity. These phenotypic effects of the mutations correlate with changes in cation uptake and are dependent on a functional Trk1-Trk2 potassium transport system. In addition, hal4 hal5 and trk1 trk2 mutants exhibit similar phenotypes: (i) they are deficient in potassium uptake; (ii) their growth is sensitive to a variety of toxic cations, including lithium, sodium, calcium, tetramethylammonium, hygromycin B, and low pH; and (iii) they exhibit increased uptake of methylammonium, an indicator of membrane potential. These results suggest that the Hal4 and Hal5 protein kinases activate the Trk1-Trk2 potassium transporter, increasing the influx of potassium and decreasing the membrane potential. The resulting loss in electrical driving force reduces the uptake of toxic cations and improves salt tolerance. Our data support a role for regulation of membrane potential in adaptation to salt stress that is mediated by the Hal4 and Hal5 kinases. PMID:10207057

  20. Protein kinase C delta (PKCδ) splice variant modulates senescence via hTERT in adipose-derived stem cells

    PubMed Central

    Carter, Gay; Patel, Rekha; Apostolatos, André; Murr, Michel; Cooper, Denise R.

    2014-01-01

    Background Adipose-derived stem cells (ADSC) were isolated and characterized from lean and obese subjects. We previously reported that distinct differences were observed in differentiating lean and obese preadipocytes. Protein kinase C delta (PKCδ) is alternatively spliced and has important roles in apoptosis. PKCδI promotes apoptosis and PKCδVIII promotes survival. Our previous data indicated an increase in the survival kinase, PKCδVIII in ADSC derived from an obese donor. We also determined that obese adipocytes were resistant to apoptosis. Here, we determine the relationship between a survival kinase PKCδVIII and hTERT expression in adipose derived stem cells from a lean and obese subject. Methods We evaluated the telomerase activity and human telomerase reverse transcriptase (hTERT) expression in lean and obese ADSC. The lean and obese ADSC were purchased as cryopreserved cells from ZenBio™ (Research Triangle Park, NC, USA). Analyses were performed using PRISM™ software and analyzed using two-tailed Student’s t-test. Results We observed an increase in telomerase in differentiating obese ADSC using western blot analysis. We determined the levels of hTERT splice variants. hTERT α+/β+ splice variant was increased after transfected of PKCδVIII. We next determined whether PKCδVIII over-expression affected the levels of telomerase. The results indicate an increase in telomerase with PKCδVIII over-expression. Conclusions Over-expression of PKCδVIII in lean ADSC substantially increased expression of hTERT and telomerase. The decreased senescence seen in obese ADSC may in part be attributed to PKCδVIII. Obese ADSC undergo lower senescence and may have increased growth potential. These results propose a larger epigenetic modification in obese ADSC compared to lean ADSC. PMID:27358850

  1. Modulation of mitogen-activated protein kinases (MAPK) activity in response to different immune stimuli in haemocytes of the common periwinkle Littorina littorea.

    PubMed

    Iakovleva, Nadya V; Gorbushin, Alexander M; Storey, Kenneth B

    2006-09-01

    The modulation of mitogen-activated protein kinase (MAPK) activity in haemocytes of the common periwinkle (Littorina littorea) in response to immune challenges by lipopolysaccharide from Echerichia coli (LPS), mannan from baker's yeast Saccharomyces cerevisiae and secretory-excretory products (SEP) of trematodes Himasthla elongata (Echinostomatidae) or after the treatment with phorbol ester (PMA) has been studied by Western blotting using affinity purified rabbit polyclonal antibodies. Exposure of the cells in suspension to PMA, LPS and mannan triggered an activation of p38 and ERK2. The JNK-mediated cascade was modulated differently by the elicitors examined. PMA treatment caused a transient activation of the JNK54 isoform, LPS exposure resulted in a decrease in activity of JNK46, and mannan had no effect on JNK phosphorylation status. Incubation of periwinkle haemocytes in culture medium containing trematode SEP did not affect the activity of any MAPK.

  2. Exchange protein activated by cAMP (Epac) mediates cAMP-dependent but protein kinase A-insensitive modulation of vascular ATP-sensitive potassium channels.

    PubMed

    Purves, Gregor I; Kamishima, Tomoko; Davies, Lowri M; Quayle, John M; Dart, Caroline

    2009-07-15

    Exchange proteins directly activated by cyclic AMP (Epacs or cAMP-GEF) represent a family of novel cAMP-binding effector proteins. The identification of Epacs and the recent development of pharmacological tools that discriminate between cAMP-mediated pathways have revealed previously unrecognized roles for cAMP that are independent of its traditional target cAMP-dependent protein kinase (PKA). Here we show that Epac exists in a complex with vascular ATP-sensitive potassium (KATP) channel subunits and that cAMP-mediated activation of Epac modulates KATP channel activity via a Ca2+-dependent mechanism involving the activation of Ca2+-sensitive protein phosphatase 2B (PP-2B, calcineurin). Application of the Epac-specific cAMP analogue 8-pCPT-2'-O-Me-cAMP, at concentrations that activate Epac but not PKA, caused a 41.6 +/- 4.7% inhibition (mean +/- S.E.M.; n = 7) of pinacidil-evoked whole-cell KATP currents recorded in isolated rat aortic smooth muscle cells. Importantly, similar results were obtained when cAMP was elevated by addition of the adenylyl cyclase activator forskolin in the presence of the structurally distinct PKA inhibitors, Rp-cAMPS or KT5720. Activation of Epac by 8-pCPT-2'-O-Me-cAMP caused a transient 171.0 +/- 18.0 nM (n = 5) increase in intracellular Ca2+ in Fura-2-loaded aortic myocytes, which persisted in the absence of extracellular Ca2+. Inclusion of the Ca2+-specific chelator BAPTA in the pipette-filling solution or preincubation with the calcineurin inhibitors, cyclosporin A or ascomycin, significantly reduced the ability of 8-pCPT-2'-O-Me-cAMP to inhibit whole-cell KATP currents. These results highlight a previously undescribed cAMP-dependent regulatory mechanism that may be essential for understanding the physiological and pathophysiological roles ascribed to arterial KATP channels in the control of vascular tone and blood flow.

  3. AKT serine/threonine protein kinase modulates baicalin-triggered autophagy in human bladder cancer T24 cells.

    PubMed

    Lin, Chingju; Tsai, Shih-Chang; Tseng, Michael T; Peng, Shu-Fen; Kuo, Sheng-Chu; Lin, Meng-Wei; Hsu, Yuan-Man; Lee, Miau-Rong; Amagaya, Sakae; Huang, Wen-Wen; Wu, Tian-Shung; Yang, Jai-Sing

    2013-03-01

    Baicalin is one of the major compounds in the traditional Chinese medicinal herb from Scutellaria baicalensis Georgi. We investigated the molecular mechanisms of cell autophagy induced by baicalin in human bladder cancer T24 cells. Baicalin inhibited cell survival as shown by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. Baicalin did not induce apoptotic cell death in T24 cells by TUNEL and caspase-3 activity assay. Baicalin induced the acidic vesicular organelle cell autophagy marker, manifested by acridine orange (AO) and monodansylcadaverine (MDC) staining and cleavage of microtubule-associated protein 1 light chain 3 (LC3). The protein expression levels of the Atg 5, Atg 7, Atg 12, Beclin-1 and LC3-II were upregulated in T24 cells after baicalin treatment. Inhibition of autophagy by 3-methyl-adenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) reduced the cleavage of LC3 in T24 cells after baicalin treatment. Furthermore, protein expression levels of phospho-AKT (Ser473) and enzyme activity of AKT were downregulated in T24 cells after baicalin treatment. In conclusion, baicalin triggered cell autophagy through the AKT signaling pathway in T24 cells.

  4. CUL3 and protein kinases

    PubMed Central

    Metzger, Thibaud; Kleiss, Charlotte; Sumara, Izabela

    2013-01-01

    Posttranslational mechanisms drive fidelity of cellular processes. Phosphorylation and ubiquitination of substrates represent very common, covalent, posttranslational modifications and are often co-regulated. Phosphorylation may play a critical role both by directly regulating E3-ubiquitin ligases and/or by ensuring specificity of the ubiquitination substrate. Importantly, many kinases are not only critical regulatory components of these pathways but also represent themselves the direct ubiquitination substrates. Recent data suggest the role of CUL3-based ligases in both proteolytic and non-proteolytic regulation of protein kinases. Our own recent study identified the mitotic kinase PLK1 as a direct target of the CUL3 E3-ligase complex containing BTB-KELCH adaptor protein KLHL22.1 In this study, we aim at gaining mechanistic insights into CUL3-mediated regulation of the substrates, in particular protein kinases, by analyzing mechanisms of interaction between KLHL22 and PLK1. We find that kinase activity of PLK1 is redundant for its targeting for CUL3-ubiquitination. Moreover, CUL3/KLHL22 may contact 2 distinct motifs within PLK1 protein, consistent with the bivalent mode of substrate targeting found in other CUL3-based complexes. We discuss these findings in the context of the existing knowledge on other protein kinases and substrates targeted by CUL3-based E3-ligases. PMID:24067371

  5. Mitogen and stress-activated protein kinase 1 (MSK1) modulates photic entrainment of the suprachiasmatic circadian clock

    PubMed Central

    Cao, Ruifeng; Butcher, Greg Q.; Karelina, Kate; Arthur, J. Simon C.; Obrietan, Karl

    2013-01-01

    The master circadian clock in mammals, the suprachiasmatic nucleus (SCN), is under the entraining influence of the external light cycle. At a mechanistic level, intracellular signaling via the p42/44 mitogen-activated protein kinase (MAPK) pathway appears to play a central role in light-evoked clock entrainment; however, the precise downstream mechanisms by which this pathway influences clock timing are not known. Within this context, we have previously reported that light stimulates activation of the MAPK effector mitogen stress activated kinase 1 (MSK1) in the SCN. In this study we utilized MSK1-/- mice to further investigate the potential role of MSK1 in circadian clock timing and entrainment. Locomotor activity analysis revealed that MSK1 null mice entrained to a 12h light/dark cycle and exhibited circadian free-running rhythms in constant darkness. Interestingly, the free running period in MSK1 null mice was significantly longer than WT control animals, and MSK1 null mice exhibited a significantly greater variance in activity onset. Further, MSK1 null mice exhibited a significant reduction in the phase delaying response to an early night light pulse (100 lux, 15 min), and, using an 8-hr phase-advancing “jet-lag” experimental paradigm MSK1 knockout animals exhibited a significantly delayed rate of re-entrainment. At the molecular level, early night light-evoked CREB phosphorylation, histone phosphorylation and Period1 gene expression were markedly attenuated in MSK1-/- animals relative to WT mice. Together, these data provide key new insights into the molecular mechanisms by which MSK1 affects the SCN clock. PMID:23127194

  6. Phospho-dependent functional modulation of GABA(B) receptors by the metabolic sensor AMP-dependent protein kinase.

    PubMed

    Kuramoto, Nobuyuki; Wilkins, Megan E; Fairfax, Benjamin P; Revilla-Sanchez, Raquel; Terunuma, Miho; Tamaki, Keisuke; Iemata, Mika; Warren, Noel; Couve, Andrés; Calver, Andrew; Horvath, Zsolt; Freeman, Katie; Carling, David; Huang, Lan; Gonzales, Cathleen; Cooper, Edward; Smart, Trevor G; Pangalos, Menelas N; Moss, Stephen J

    2007-01-18

    GABA(B) receptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly rectifying K(+) channels (GIRKs) and inhibiting Ca(2+) channels. We demonstrate here that GABA(B) receptors are intimately associated with 5'AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5'AMP concentration that are caused by enhanced metabolic activity, anoxia, or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABA(B) receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally, we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a neuroprotective mechanism, which, under conditions of metabolic stress or after ischemia, increases GABA(B) receptor function to reduce excitotoxicity and thereby promotes neuronal survival.

  7. Phospho-dependent functional modulation of GABAB receptors by the metabolic sensor AMP-dependent protein kinase

    PubMed Central

    Kuramoto, Nobuyuki; Wilkins, Megan E; Fairfax, Benjamin P; Revilla-Sanchez, Raquel; Terunuma, Miho; Warren, Noel; Tamaki, Keisuke; Iemata, Mika; Couve, Andrés; Calver, Andrew; Horvath, Zsolt; Freeman, Katie; Carling, David; Huang, Lan; Gonzales, Cathleen; Cooper, Edward; Smart, Trevor G.; Pangalos, Menelas N.; Moss., Stephen J.

    2007-01-01

    GABAB receptors are heterodimeric G-protein coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly-rectifying K+ channels (GIRKs) and inhibiting Ca2+ channels. We demonstrate here that GABAB receptors are intimately associated with 5’AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5’AMP concentration caused by enhanced metabolic activity, anoxia or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABAB receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a novel neuroprotective mechanism, which under conditions of metabolic stress or after ischemia increases GABAB receptor function to reduce excitotoxicity and thereby promoting neuronal survival. PMID:17224405

  8. A-kinase Anchoring Protein 79/150 Recruits Protein Kinase C to Phosphorylate Roundabout Receptors.

    PubMed

    Samelson, Bret K; Gore, Bryan B; Whiting, Jennifer L; Nygren, Patrick J; Purkey, Alicia M; Colledge, Marcie; Langeberg, Lorene K; Dell'Acqua, Mark L; Zweifel, Larry S; Scott, John D

    2015-05-29

    Anchoring proteins direct protein kinases and phosphoprotein phosphatases toward selected substrates to control the efficacy, context, and duration of neuronal phosphorylation events. The A-kinase anchoring protein AKAP79/150 interacts with protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (calcineurin) to modulate second messenger signaling events. In a mass spectrometry-based screen for additional AKAP79/150 binding partners, we have identified the Roundabout axonal guidance receptor Robo2 and its ligands Slit2 and Slit3. Biochemical and cellular approaches confirm that a linear sequence located in the cytoplasmic tail of Robo2 (residues 991-1070) interfaces directly with sites on the anchoring protein. Parallel studies show that AKAP79/150 interacts with the Robo3 receptor in a similar manner. Immunofluorescent staining detects overlapping expression patterns for murine AKAP150, Robo2, and Robo3 in a variety of brain regions, including hippocampal region CA1 and the islands of Calleja. In vitro kinase assays, peptide spot array mapping, and proximity ligation assay staining approaches establish that human AKAP79-anchored PKC selectively phosphorylates the Robo3.1 receptor subtype on serine 1330. These findings imply that anchored PKC locally modulates the phosphorylation status of Robo3.1 in brain regions governing learning and memory and reward.

  9. Mediator kinase module and human tumorigenesis

    PubMed Central

    Clark, Alison D.; Oldenbroek, Marieke; Boyer, Thomas G.

    2016-01-01

    Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit “kinase” module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways. PMID:26182352

  10. Roles of receptor for activated protein kinase C1 for modulating immune responses in white shrimp Litopenaeus vannamei.

    PubMed

    Chang, Zhong-Wen; Chang, Chin-Chyuan

    2015-10-01

    Complementary (c)DNA encoding a receptor for activated protein kinase C1 (RACK1) messenger (m)RNA of the white shrimp Litopenaeus vannamei, designated LvRACK1, consisted a 1136-bp cDNA containing an open reading frame (ORF) of 954 bp, a 111-bp 5'-untranslated region (UTR), and a 71-bp 3'-UTR, which is a 36 kDa cytosolic protein, belonging to the Trp-Asp40 (WD40) family of proteins, characterized by containing seven highly conserved Trp-Asp40 (WD40) internal repeats, and a poly A tail. The WD repeat of LvRACK1 can be predicted to form a seven-bladed propeller structure with each WD repeat composed of four antiparallel β-sheets. The WD40 domains have been implicated in protein-protein interactions. A comparison of amino acid sequences showed that LvRACK1 was closely related to arthropods RACK1. LvRACK1 cDNA was synthesized in all tested tissues detected with real-time PCR including haemocytes, hepatopancreas, gills, muscles, subcuticular epithelium, intestines, abdominal nervous ganglia, thoracic nervous ganglia, lymphoid organ, stomach, heart, and antennal gland, especially in subcuticular epithelium and gill. LvRACK1 mRNA transcription in haemocytes of L. vannamei injected with Vibrio alginolyticus decreased. The depletion of LvRACK1 of haemocytes in L. vannamei received its dsRNA revealed the increased respiratory bursts per haemocyte, superoxide dismutase (SOD), activity, glutathione peroxidase (GPx) activity, and clotting time, but showed the decreased total haemocyte count (THC), hyaline cells (HCs), phagocytic activity, and transglutaminase (TG) activity. LvRACK1 silenced shrimp showed the upregulated gene expressions of cyMnSOD, mtMnSOD, peroxinectin (PE), and TGI, and showed the downregulated α2-macroglobulin (α2-M), clottable protein (CP), lysozyme, and crustin gene expressions. It is therefore concluded that LvRACK1 is involved in immune defense and signaling transduction in haemocytes of L. vannamei infected with V. alginolyticus.

  11. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1999-01-01

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

  12. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

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

  13. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Lin, Anning

    1999-11-30

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

  14. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2004-03-16

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

  15. Oncoprotein protein kinase

    DOEpatents

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

    2003-02-04

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

  16. Oncoprotein protein kinase

    DOEpatents

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

    2005-03-08

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

  17. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

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

  18. Oncoprotein protein kinase

    DOEpatents

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

    2005-01-25

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

  19. Oncoprotein protein kinase

    DOEpatents

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

    1997-02-25

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

  20. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1998-01-01

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

  1. Phosphorylation of the cAMP-dependent protein kinase (PKA) regulatory subunit modulates PKA-AKAP interaction, substrate phosphorylation, and calcium signaling in cardiac cells.

    PubMed

    Manni, Sabrina; Mauban, Joseph H; Ward, Christopher W; Bond, Meredith

    2008-08-29

    Subcellular compartmentalization of the cAMP-dependent protein kinase (PKA) by protein kinase A-anchoring proteins (AKAPs) facilitates local protein phosphorylation. However, little is known about how PKA targeting to AKAPs is regulated in the intact cell. PKA binds to an amphipathic helical region of AKAPs via an N-terminal domain of the regulatory subunit. In vitro studies showed that autophosphorylation of type II regulatory subunit (RII) can alter its affinity for AKAPs and the catalytic subunit (PKA(cat)). We now investigate whether phosphorylation of serine 96 on RII regulates PKA targeting to AKAPs, downstream substrate phosphorylation and calcium cycling in primary cultured cardiomyocytes. We demonstrated that, whereas there is basal phosphorylation of RII subunits, persistent maximal activation of PKA results in a phosphatase-dependent loss of RII phosphorylation. To investigate the functional effects of RII phosphorylation, we constructed adenoviral vectors incorporating mutants which mimic phosphorylated (RIIS96D), nonphosphorylated (RIIS96A) RII, or wild-type (WT) RII and performed adenoviral infection of neonatal rat cardiomyocytes. Coimmunoprecipitation showed that more AKAP15/18 was pulled down by the phosphomimic, RIIS96D, than RIIS96A. Phosphorylation of phospholamban and ryanodine receptor was significantly increased in cells expressing RIIS96D versus RIIS96A. Expression of recombinant RII constructs showed significant effects on cytosolic calcium transients. We propose a model illustrating a central role of RII phosphorylation in the regulation of local PKA activity. We conclude that RII phosphorylation regulates PKA-dependent substrate phosphorylation and may have significant implications for modulation of cardiac function.

  2. Heat-shock protein 70 modulates apoptosis signal-regulating kinase 1 in stressed hepatocytes of Mugil cephalus.

    PubMed

    Padmini, Ekambaram; Tharani, Jayachandran

    2014-10-01

    Oxidative stress causes damage at the cellular level and activates a number of signaling pathways. Heat-shock proteins (HSPs) play an important role in repair and protective mechanisms under cell response to stress conditions. HSP70 has been shown to act as an inhibitor of apoptosis. Apoptosis signal-regulating kinase-1 (ASK1) activity is regulated at multiple levels, one of which is through inhibition by cytosolic chaperons HSP70. The current study was aimed to investigate the alteration in signaling molecules that allow the fish to survive under stressed natural field conditions. The study also investigates the variation in biomolecular composition of hepatocytes by using Fourier transform infrared spectroscopy. The impact of stress on hepatocytes was assessed by measuring the level of lipid peroxides (LPO), catalase activity (CAT) and assessing the changes in hepatocytes of Mugil cephalus inhabiting Kovalam and Ennore estuaries. The expression of HSP70 and ASK1 were analyzed by immunoblot analysis and ELISA, respectively. The spectral analysis showed variations in biomolecular composition of hepatocytes at a wave number region of 4,000-400 cm(-1). There was significant decrease of CAT activity (p < 0.01) (25 %) with significant increase of LPO (p < 0.001) (35 %) and HSP70 (p < 0.001) and insignificant increase of ASK1 (p < 0.05) (16 %) in fish hepatocytes inhabiting Ennore estuary than Kovalam estuary. In conclusion, the present study suggests that the survival of fish in the Ennore estuary under stressed condition may be due to the upregulation of HSP70 that mediates the altered signal pathway which promotes cellular resistance against apoptosis.

  3. Calcium- and guanine-nucleotide-dependent exocytosis in permeabilized rat mast cells. Modulation by protein kinase C.

    PubMed Central

    Koopmann, W R; Jackson, R C

    1990-01-01

    We have used a digitonin-permeabilized cell system to study the signal transduction pathways responsible for stimulus-secretion coupling in the rat peritoneal mast cell. Conditions were established for permeabilizing the mast cell plasma membrane without disrupting secretory vesicles. Exocytotic release of histamine from digitonin-permeabilized cells required a combination of micromolar concentrations of Ca2+ and the stable guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]), but was independent of exogenous ATP. In the presence of 40 microM-GTP[S], exocytosis was half-maximal at 1.3 microM-Ca2+ and maximal at 10 microM-Ca2+; GTP[S] alone (100 microM) had no effect on histamine release in the absence of added Ca2+. In the presence of 10 microM free Ca2+, 5 microM-GTP[S] was required for half-maximal exocytosis. To examine the possible role of protein kinase C (PKC) in exocytosis, we utilized 12-O-tetradecanoylphorbol 13-acetate (TPA) to activate PKC and studied its effect on histamine release from permeabilized mast cells. Cells that had been incubated with TPA (25 nM for 5 min) exhibited increased sensitivity to both GTP[S] and Ca2+. The PKC inhibitor staurosporine blocked the effect of TPA without inhibiting normal exocytosis in response to the combination of GTP[S] and Ca2+. In addition, down-regulation of mast-cell PKC by long-term TPA treatment (25 nM for 20 h) blocked the ability of the cells to respond to TPA and inhibited exocytosis in response to Ca2+ and GTP[S] by 40-50%. These results suggest that the sensitivity of the exocytotic machinery of the mast cell can be altered by PKC-catalysed phosphorylation events, but that activation of PKC is not required for exocytosis to occur. Images Fig. 7. PMID:1689146

  4. Differential modulation of evoked and spontaneous glycine release from rat spinal cord glycinergic terminals by the cyclic AMP/protein kinase A transduction cascade.

    PubMed

    Katsurabayashi, Shutaro; Kubota, Hisahiko; Moorhouse, Andrew J; Akaike, Norio

    2004-11-01

    The mechanisms underlying cyclic AMP modulation of action potential-dependent and -independent (spontaneous) release of glycine from terminals synapsing onto sacral dorsal commissural nucleus neurons of lamina X were studied in spinal cord slices using conventional patch-clamp recordings. 3-Isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, and forskolin increased the amplitude of evoked inhibitory postsynaptic currents (eIPSCs) in a sensitive manner to protein kinase A (PKA) inhibition (with KT-5720). Direct activation (with adenosine 3',5'-cyclic-monophosphothioate, Sp-isomer) and inhibition (with adenosine 3',5'-cyclic-monophosphothioate, Rp-isomer) of PKA increased and decreased the eIPSC amplitude, respectively. Paired pulse experiments and direct injection of PKA inhibitor fragment 6-22 amide (PKI(6-22)) into the recording neuron revealed that these effects on eIPSC amplitude occurred presynaptically, indicating that evoked glycine release is regulated by presynaptic cAMP via changes in PKA activity. Increasing cAMP also increased spontaneous release of glycine, causing an increased frequency of miniature IPSCs (mIPSCs). In contrast to the effects on evoked release, this response was not solely mediated via PKA, as it was not occluded by PKA inhibition, and both direct inhibition and direct activation of PKA actually enhanced mIPSC frequency. Direct inhibition of cAMP (with SQ 22536) did, however, reduce mIPSC frequency. These results suggest cAMP modulation of evoked and spontaneous release involves different presynaptic mechanisms and proteins.

  5. Modulation of Spc1 stress-activated protein kinase activity by methylglyoxal through inhibition of protein phosphatase in the fission yeast Schizosaccharomyces pombe

    SciTech Connect

    Takatsume, Yoshifumi; Izawa, Shingo; Inoue, Yoshiharu

    2007-11-30

    Methylglyoxal, a ubiquitous metabolite derived from glycolysis has diverse physiological functions in yeast cells. Previously, we have reported that extracellularly added methylglyoxal activates Spc1, a stress-activated protein kinase (SAPK), in the fission yeast Schizosaccharomyces pombe [Y. Takatsume, S. Izawa, Y. Inoue, J. Biol. Chem. 281 (2006) 9086-9092]. Phosphorylation of Spc1 by treatment with methylglyoxal in S. pombe cells defective in glyoxalase I, an enzyme crucial for the metabolism of methylglyoxal, continues for a longer period than in wild-type cells. Here we show that methylglyoxal inhibits the activity of the protein phosphatase responsible for the dephosphorylation of Spc1 in vitro. In addition, we found that methylglyoxal inhibits human protein tyrosine phosphatase 1B (PTP1B) also. We propose a model for the regulation of the activity of the Spc1-SAPK signaling pathway by methylglyoxal in S. pombe.

  6. Baicalin attenuates high fat diet-induced insulin resistance and ectopic fat storage in skeletal muscle, through modulating the protein kinase B/Glycogen synthase kinase 3 beta pathway.

    PubMed

    Xi, You-Li; Li, Hong-Xia; Chen, Chen; Liu, Ya-Qun; Lv, Hong-Mei; Dong, Shi-Qi; Luo, Er-Fei; Gu, Ming-Bo; Liu, Hua

    2016-01-01

    Insulin resistance is the pathophysiological basis of many diseases. Overcoming early insulin resistance highly significant in prevention diabetes, non-alcoholic fatty liver, and atherosclerosis. The present study aimed at evaluating the therapeutic effects of baicalin on insulin resistance and skeletal muscle ectopic fat storage in high fat diet-induced mice, and exploring the potential molecular mechanisms. Insulin resistance in mice was induced with a high fat diet for 16 weeks. Animals were then treated with three different doses of baicalin (100, 200, and 400 mg·kg(-1)·d(-1)) for 14 weeks. Fasting blood glucose, fasting serum insulin, glucose tolerance test (GTT), insulin tolerance test (ITT), and skeletal muscle lipid deposition were measured. Additionally, the AMP-activated protein kinase/acetyl-CoA carboxylase and protein kinase B/Glycogen synthase kinase 3 beta pathways in skeletal muscle were further evaluated. Baicalin significantly reduced the levels of fasting blood glucose and fasting serum insulin and attenuated high fat diet induced glucose tolerance and insulin tolerance. Moreover, insulin resistance was significantly reversed. Pathological analysis revealed baicalin dose-dependently decreased the degree of the ectopic fat storage in skeletal muscle. The properties of baicalin were mediated, at least in part, by inhibition of the AMPK/ACC pathway, a key regulator of de novo lipogenesis and activation of the Akt/GSK-3β pathway, a key regulator of Glycogen synthesis. These data suggest that baicalin, at dose up to 400 mg·kg(-1)·d(-1), is safe and able to attenuate insulin resistance and skeletal muscle ectopic fat storage, through modulating the skeletal muscle AMPK/ACC pathway and Akt/GSK-3β pathway.

  7. Differential modulation of CYP2E1 activity by cAMP-dependent protein kinase upon Ser129 replacement.

    PubMed

    Oesch-Bartlomowicz, B; Padma, P R; Becker, R; Richter, B; Hengstler, J G; Freeman, J E; Wolf, C R; Oesch, F

    1998-07-10

    Many toxic compounds are activated by cytochrome P450 (CYP) 2E1 to reactive metabolites, which represents a potential hazard for cellular homeostasis. Therefore knowledge about CYP2E1 regulation could be of great biological importance. It has been shown that CYP2E1 is controlled transcriptionally and post-translationally by phosphorylation. In the present study we investigated the role of serine-129 (Ser129) in the protein kinase A (PKA) recognition sequence motif Arg-Arg-Phe-Ser129. To gain further insights into the possible relevance of Ser129 for CYP2E1 function, Ser129 was replaced by alanine (Ala) or glycine (Gly) by site-directed mutations of the cDNA coding for CYP2E1. The mutant cDNAs were transfected into Chinese hamster lung fibroblast V79 cells. Despite the mutation in the PKA phosphorylation motif, all strains produced catalytically active CYP2E1. However, there was a marked change in the substrate preference: The Gly129-containing strains hydroxylated p-nitrophenol (PNP) to a markedly higher extent than the wild-type cDNA-containing cells, while they demethylated N-nitrosodimethylamine (NDMA) to a markedly lower extent than the wild-type cells. All the strains activated NDMA to mutagenic products. Treatment with the membrane-permeating cAMP derivative db-cAMP reduced markedly both the PNP hydroxylase and the NDMA demethylase activities as well as the mutation frequency induced by NDMA in the Ser129-containing strain. This decrease in activity was not accompanied by a decrease in CYP2E1 content. In addition, the catalytic activities of CYP2E1 were decreased in microsomes from rat hepatocytes treated with db-cAMP. Also in this case, the decrease in activities was not accompanied by a decrease in enzyme protein. These findings argue that involvement of Ser129 and its phosphorylation is not in determining CYP2E1 protein level, but rather in controlling its catalytic activity. In contrast, in the strains containing Ala129 or Gly129, treatment with db

  8. Mitogen-activated protein kinase cascades in Vitis vinifera

    PubMed Central

    Çakır, Birsen; Kılıçkaya, Ozan

    2015-01-01

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

  9. Ubiquitination mediates Kv1.3 endocytosis as a mechanism for protein kinase C-dependent modulation.

    PubMed

    Martínez-Mármol, Ramón; Styrczewska, Katarzyna; Pérez-Verdaguer, Mireia; Vallejo-Gracia, Albert; Comes, Núria; Sorkin, Alexander; Felipe, Antonio

    2017-02-10

    The voltage-dependent potassium channel Kv1.3 plays essential physiological functions in the immune system. Kv1.3, regulating the membrane potential, facilitates downstream Ca(2+) -dependent pathways and becomes concentrated in specific membrane microdomains that serve as signaling platforms. Increased and/or delocalized expression of the channel is observed at the onset of several autoimmune diseases. In this work, we show that adenosine (ADO), which is a potent endogenous modulator, stimulates PKC, thereby causing immunosuppression. PKC activation triggers down-regulation of Kv1.3 by inducing a clathrin-mediated endocytic event that targets the channel to lysosomal-degradative compartments. Therefore, the abundance of Kv1.3 at the cell surface decreases, which is clearly compatible with an effective anti-inflammatory response. This mechanism requires ubiquitination of Kv1.3, catalyzed by the E3 ubiquitin-ligase Nedd4-2. Postsynaptic density protein 95 (PSD-95), a member of the MAGUK family, recruits Kv1.3 into lipid-raft microdomains and protects the channel against ubiquitination and endocytosis. Therefore, the Kv1.3/PSD-95 association fine-tunes the anti-inflammatory response in leukocytes. Because Kv1.3 is a promising multi-therapeutic target against human pathologies, our results have physiological relevance. In addition, this work elucidates the ADO-dependent PKC-mediated molecular mechanism that triggers immunomodulation by targeting Kv1.3 in leukocytes.

  10. Ubiquitination mediates Kv1.3 endocytosis as a mechanism for protein kinase C-dependent modulation

    PubMed Central

    Martínez-Mármol, Ramón; Styrczewska, Katarzyna; Pérez-Verdaguer, Mireia; Vallejo-Gracia, Albert; Comes, Núria; Sorkin, Alexander; Felipe, Antonio

    2017-01-01

    The voltage-dependent potassium channel Kv1.3 plays essential physiological functions in the immune system. Kv1.3, regulating the membrane potential, facilitates downstream Ca2+ -dependent pathways and becomes concentrated in specific membrane microdomains that serve as signaling platforms. Increased and/or delocalized expression of the channel is observed at the onset of several autoimmune diseases. In this work, we show that adenosine (ADO), which is a potent endogenous modulator, stimulates PKC, thereby causing immunosuppression. PKC activation triggers down-regulation of Kv1.3 by inducing a clathrin-mediated endocytic event that targets the channel to lysosomal-degradative compartments. Therefore, the abundance of Kv1.3 at the cell surface decreases, which is clearly compatible with an effective anti-inflammatory response. This mechanism requires ubiquitination of Kv1.3, catalyzed by the E3 ubiquitin-ligase Nedd4-2. Postsynaptic density protein 95 (PSD-95), a member of the MAGUK family, recruits Kv1.3 into lipid-raft microdomains and protects the channel against ubiquitination and endocytosis. Therefore, the Kv1.3/PSD-95 association fine-tunes the anti-inflammatory response in leukocytes. Because Kv1.3 is a promising multi-therapeutic target against human pathologies, our results have physiological relevance. In addition, this work elucidates the ADO-dependent PKC-mediated molecular mechanism that triggers immunomodulation by targeting Kv1.3 in leukocytes. PMID:28186199

  11. 5-HT1A receptor-mediated phosphorylation of extracellular signal-regulated kinases (ERK1/2) is modulated by regulator of G protein signaling protein 19.

    PubMed

    Wang, Qin; Terauchi, Akiko; Yee, Christopher H; Umemori, Hisashi; Traynor, John R

    2014-09-01

    The 5-HT1A receptor is a G protein coupled receptor (GPCR) that activates G proteins of the Gαi/o family. 5-HT1A receptors expressed in the raphe, hippocampus and prefrontal cortex are implicated in the control of mood and are targets for anti-depressant drugs. Regulators of G protein signaling (RGS) proteins are members of a large family that play important roles in signal transduction downstream of G protein coupled receptors (GPCRs). The main role of RGS proteins is to act as GTPase accelerating proteins (GAPs) to dampen or negatively regulate GPCR-mediated signaling. We have shown that a mouse expressing Gαi2 that is insensitive to all RGS protein GAP activity has an anti-depressant-like phenotype due to increased signaling of postsynaptic 5-HT1A receptors, thus implicating the 5-HT1A receptor-Gαi2 complex as an important target. Here we confirm that RGS proteins act as GAPs to regulate signaling to adenylate cyclase and the mitogen-activated protein kinase (MAPK) pathway downstream of the 5-HT1A receptor, using RGS-insensitive Gαi2 protein expressed in C6 cells. We go on to use short hairpin RNA (shRNA) to show that RGS19 is responsible for the GAP activity in C6 cells and also that RGS19 acts as a GAP for 5-HT1A receptor signaling in human neuroblastoma SH-SY5Y cells and primary hippocampal neurons. In addition, in both cell types the synergy between 5-HT1A receptor and the fibroblast growth factor receptor 1 in stimulating the MAPK pathway is enhanced following shRNA reduction of RGS19 expression. Thus RGS19 may be a viable new target for anti-depressant medications.

  12. Activation of protein kinase C as a modulator of potentiated UK-14304-induced contractions in dog mesenteric artery and vein.

    PubMed

    Shimamoto, H; Shimamoto, Y; Kwan, C Y; Daniel, E E

    1995-12-01

    We assessed the role of protein kinase C (PKC) in the mechanism responsible for the potentiation of UK-14304-induced contractions produced when isolated dog mesenteric vascular rings were pretreated with threshold concentrations of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), KCl, or endothelin-1 (ET-1). In dog mesenteric artery. UK-14304 produced a biphasic concentration-response curve in the presence of TPA, KCl, or ET-1, with the curve portion at lower concentrations being alpha 2-adrenoceptor dependent and the portion at higher concentrations being alpha 1-adrenoceptor dependent. Calphostin C (10(-6)M), a PKC inhibitor, abolished amplified UK-14304-induced contraction in the TPA-pretreated tissues. In the KCl- and ET-1-pretreated tissues. 10(-6)M calphostin C antagonized amplified UK-14304-induced contractions by approximately 20% in both parts of the concentration-response curve. In contrast, in dog mesenteric vein, amplified UK-14304-induced contractions by TPA, KCl, and ET-1 were entirely dependent on alpha 2-adrenoceptors. Calphostin C (10(-6)M), which in control experiments had no effect on KCl-induced contraction and antagonized responses to TPA by 60.1%, inhibited UK-14304-induced contraction by 18.3%. Amplified UK-14304-induced contraction was antagonized by 10(-6)M calphostin C by 21.8% in KCl-precontracted tissues, 58.1% in ET-1-precontracted tissues, and 66.3% in TPA-precontracted tissues. In the ET-1- and TPA-pretreated dog mesenteric veins, 10(-6)M calphostin C decreased maximal tensions of enhanced UK-14304-induced contractions to the same level as the UK-14304-induced maximal tension inhibited by 10(-6)M calphostin C in untreated dog mesenteric vein. Therefore, TPA can be a precontracting agent that amplifies UK-14304-induced contractions through PKC activation in both dog mesenteric artery and vein. PKC predominantly mediates the contraction amplification mechanisms after exposure to ET-1 in dog mesenteric vein and does not play a major role in

  13. Modulation of Kv3.4 channel N-type inactivation by protein kinase C shapes the action potential in dorsal root ganglion neurons.

    PubMed

    Ritter, David M; Ho, Cojen; O'Leary, Michael E; Covarrubias, Manuel

    2012-01-01

    Fast inactivation of heterologously expressed Kv3.4 channels is dramatically slowed upon phosphorylation of the channel's N-terminal (N-type) inactivation gate by protein kinase C (PKC). However, the presence and physiological importance of this exquisite modulation in excitable tissues were unknown. Here, we employed minimally invasive cell-attached patch-clamping, single-cell qPCR and specific siRNAs to unambiguously demonstrate that fast-inactivating Kv3.4 channels underlie a robust high voltage-activated A-type K(+) current (I(AHV)) in nociceptive dorsal root ganglion neurons from 7-day-old rats. We also show that PKC activation with phorbol 12,13-dibutyrate (PDBu) causes a 4-fold slowing of Kv3.4 channel inactivation and, consequently, accelerates the repolarization of the action potential (AP) by 22%, which shortens the AP duration by 14%. G-protein coupled receptor (GPCR) agonists eliminate I(AHV) fast inactivation in a membrane-delimited manner, suggesting a Kv3.4 channel signalling complex. Preincubation of the neurons with the PKC inhibitor bisindolylmaleimide II inhibits the effect of GPCR agonists and PDBu. Furthermore, activation of PKC via GPCR agonists recapitulates the effects of PDBu on the AP. Finally, transfection of the neurons with Kv3.4 siRNA prolongs the AP by 25% and abolishes the GPCR agonist-induced acceleration of the AP repolarization. These results show that Kv3.4 channels help shape the repolarization of the nociceptor AP, and that modulation of Kv3.4 channel N-type inactivation by PKC regulates AP repolarization and duration. We propose that the dramatic modulation of I(AHV) fast inactivation by PKC represents a novel mechanism of neural plasticity with potentially significant implications in the transition from acute to chronic pain.

  14. Degradation of Activated Protein Kinases by Ubiquitination

    PubMed Central

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases. PMID:19489726

  15. The Rice Transcription Factor WRKY53 Suppresses Herbivore-Induced Defenses by Acting as a Negative Feedback Modulator of Mitogen-Activated Protein Kinase Activity1

    PubMed Central

    Hu, Lingfei; Ye, Meng; Zhang, Tongfang; Zhou, Guoxin; Wang, Qi; Lu, Jing

    2015-01-01

    The mechanisms by which herbivore-attacked plants activate their defenses are well studied. By contrast, little is known about the regulatory mechanisms that allow them to control their defensive investment and avoid a defensive overshoot. We characterized a rice (Oryza sativa) WRKY gene, OsWRKY53, whose expression is rapidly induced upon wounding and induced in a delayed fashion upon attack by the striped stem borer (SSB) Chilo suppressalis. The transcript levels of OsWRKY53 are independent of endogenous jasmonic acid but positively regulated by the mitogen-activated protein kinases OsMPK3/OsMPK6. OsWRKY53 physically interacts with OsMPK3/OsMPK6 and suppresses their activity in vitro. By consequence, it modulates the expression of defensive, MPK-regulated WRKYs and thereby reduces jasmonic acid, jasmonoyl-isoleucine, and ethylene induction. This phytohormonal reconfiguration is associated with a reduction in trypsin protease inhibitor activity and improved SSB performance. OsWRKY53 is also shown to be a negative regulator of plant growth. Taken together, these results show that OsWRKY53 functions as a negative feedback modulator of MPK3/MPK6 and thereby acts as an early suppressor of induced defenses. OsWRKY53 therefore enables rice plants to control the magnitude of their defensive investment during early signaling. PMID:26453434

  16. p-Coumaric acid modulates glucose and lipid metabolism via AMP-activated protein kinase in L6 skeletal muscle cells.

    PubMed

    Yoon, Seon-A; Kang, Seong-Il; Shin, Hye-Sun; Kang, Seung-Woo; Kim, Jeong-Hwan; Ko, Hee-Chul; Kim, Se-Jae

    2013-03-22

    p-Coumaric acid (3-[4-hydroxyphenyl]-2-propenoic acid) is a ubiquitous plant metabolite with antioxidant, anti-inflammatory, and anticancer properties. In this study, we examined whether p-coumaric acid modulates glucose and lipid metabolism via AMP-activated protein kinase (AMPK) in L6 skeletal muscle cells. p-Coumaric acid increased the phosphorylation of AMPK in a dose-dependent manner in differentiated L6 skeletal muscle cells. It also increased the phosphorylation of acetyl-CoA carboxylase (ACC) and the expression of CPT-1 mRNA and PPARα, suggesting that it promotes the β-oxidation of fatty acids. Also, it suppressed oleic acid-induced triglyceride accumulation, and enhanced 2-NBDG uptake in differentiated L6 muscle cells. Pretreatment with compound C inhibited AMPK activation, reduced ACC phosphorylation and 2-NBDG uptake, and increased triglyceride accumulation. However, p-coumaric acid counterbalanced the inhibitory effects of compound C. Taken together, these results suggest that p-coumaric acid modulates glucose and lipid metabolism via AMPK activation in L6 skeletal muscle cells and that it has potentially beneficial effects in improving or treating metabolic disorders.

  17. Inverse modulation of the energy sensor Snf1-related protein kinase 1 on hypoxia adaptation and salt stress tolerance in Arabidopsis thaliana.

    PubMed

    Im, Jong Hee; Cho, Young-Hee; Kim, Geun-Don; Kang, Geun-Ho; Hong, Jung-Woo; Yoo, Sang-Dong

    2014-10-01

    Terrestrial plants are exposed to complex stresses of high salt-induced abscisic acid (ABA) and submergence-induced hypoxia when seawater floods fields. Many studies have investigated plant responses to individual stress conditions, but not so much for coupled or sequentially imposed stresses. We examined molecular regulatory mechanisms of gene expression underlying the cellular responses involved in crosstalk between salt and hypoxia stresses. Salt/ABA- and AtMYC2-dependent induction of a synthetic ABA-responsive element and the native RD22 promoters were utilized in our cell-based functional assays. Such promoter-based reporter induction was largely inhibited by hypoxia and hypoxia-inducible AKIN10 activity. Biochemical analyses showed that AKIN10 negatively modulates AtMYC2 protein accumulation via proteasome activity upon AKIN10 kinase activity-dependent protein modification. Further genetic analysis using transgenic plants expressing AKIN10 provided evidence that AKIN10 activity undermined AtMYC2-dependent salt tolerance. Our findings unravel a novel molecular interaction between the key signalling constituents leading crosstalk between salt and hypoxia stresses in Arabidopsis thaliana under the detrimental condition of submergence in saltwater.

  18. IMPACT is a developmentally regulated protein in neurons that opposes the eukaryotic initiation factor 2α kinase GCN2 in the modulation of neurite outgrowth.

    PubMed

    Roffé, Martín; Hajj, Glaucia N M; Azevedo, Hátylas F; Alves, Viviane S; Castilho, Beatriz A

    2013-04-12

    The product of the mouse Imprinted and Ancient gene, IMPACT, is preferentially expressed in neurons. We have previously shown that IMPACT overexpression inhibits the activation of the protein kinase GCN2, which signals amino acid starvation. GCN2 phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in inhibition of general protein synthesis but increased translation of specific messages, such as ATF4. GCN2 is also involved in the regulation of neuronal functions, controlling synaptic plasticity, memory, and feeding behavior. We show here that IMPACT abundance increases during differentiation of neurons and neuron-like N2a cells, whereas GCN2 displays lowered activation levels. Upon differentiation, IMPACT associates with translating ribosomes, enhances translation initiation, and down-regulates the expression of ATF4. We further show that endogenous IMPACT promotes neurite outgrowth whereas GCN2 is a strong inhibitor of spontaneous neuritogenesis. Together, these results uncover the participation of the GCN2-IMPACT module of translational regulation in a highly controlled step in the development of the nervous system.

  19. Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae.

    PubMed Central

    Mösch, H U; Roberts, R L; Fink, G R

    1996-01-01

    RAS2val19, a dominant activated form of Saccharomyces cerevisiae Ras2, stimulates both filamentous growth and expression of a transcriptional reporter FG(TyA)::lacZ but does not induce the mating pathway reporter FUS1::lacZ. This induction depends upon elements of the conserved mitogen-activated protein kinase (MAPK) pathway that is required for both filamentous growth and mating, two distinct morphogenetic events. Full induction requires Ste20 (homolog of mammalian p65PAK protein kinases), Ste11 [an MEK kinase (MEKK) or MAPK kinase (MEK) kinase], Ste7 (MEK or MAPK kinase), and the transcription factor Ste12. Moreover, the Rho family protein Cdc42, a conserved morphogenetic G protein, is also a potent regulator of filamentous growth and FG(TyA)::lacZ expression in S. cerevisiae. Stimulation of both filamentous growth and FG(TyA)::lacZ by Cdc42 depends upon Ste20. In addition, dominant negative CDC42Ala118 blocks RAS2val19 activation, placing Cdc42 downstream of Ras2. Our results suggest that filamentous growth in budding yeast is regulated by an evolutionarily conserved signaling pathway that controls cell morphology. Images Fig. 1 Fig. 2 Fig. 3 PMID:8643578

  20. c-Jun N-terminal kinase (JNK)-mediated modulation of brain mitochondria function: new target proteins for JNK signalling in mitochondrion-dependent apoptosis.

    PubMed Central

    Schroeter, Hagen; Boyd, Clinton S; Ahmed, Ruhi; Spencer, Jeremy P E; Duncan, Roger F; Rice-Evans, Catherine; Cadenas, Enrique

    2003-01-01

    The molecular mechanisms underlying the initiation and control of the release of cytochrome c during mitochondrion-dependent apoptosis are thought to involve the phosphorylation of mitochondrial Bcl-2 and Bcl-x(L). Although the c-Jun N-terminal kinase (JNK) has been proposed to mediate the phosphorylation of Bcl-2/Bcl-x(L) the mechanisms linking the modification of these proteins and the release of cytochrome c remain to be elucidated. This study was aimed at establishing interdependency between JNK signalling and mitochondrial apoptosis. Using an experimental model consisting of isolated, bioenergetically competent rat brain mitochondria, these studies show that (i) JNK catalysed the phosphorylation of Bcl-2 and Bcl-x(L) as well as other mitochondrial proteins, as shown by two-dimensional isoelectric focusing/SDS/PAGE; (ii) JNK-induced cytochrome c release, in a process independent of the permeability transition of the inner mitochondrial membrane (imPT) and insensitive to cyclosporin A; (iii) JNK mediated a partial collapse of the mitochondrial inner-membrane potential (Deltapsim) in an imPT- and cyclosporin A-independent manner; and (iv) JNK was unable to induce imPT/swelling and did not act as a co-inducer, but as an inhibitor of Ca-induced imPT. The results are discussed with regard to the functional link between the Deltapsim and factors influencing the permeability transition of the inner and outer mitochondrial membranes. Taken together, JNK-dependent phosphorylation of mitochondrial proteins including, but not limited to, Bcl-2/Bcl-x(L) may represent a potential of the modulation of mitochondrial function during apoptosis. PMID:12614194

  1. Epigenetic modulation of the protein kinase A RIIα (PRKAR2A) gene by histone deacetylases 1 and 2 in human smooth muscle cells.

    PubMed

    Karolczak-Bayatti, Magdalena; Loughney, Andrew D; Robson, Stephen C; Europe-Finner, G Nicholas

    2011-01-01

    Recently we reported that the expression of the protein kinase A (PKA) regulatory subunit RIIα is dynamically regulated in human smooth muscle cells of the uterus. We showed that expression levels of mRNA/protein were substantially increased during pregnancy and decreased upon labour, changes that were mirrored by particulate type II PKA activity. This implied an important role for RIIα in maintaining uterine quiescence during pregnancy. Consequently the purpose of the present study was to identify potential mechanisms by which expression of the RIIα gene was regulated in this tissue. We indicate here that the three SpI-III (GC) binding domains within the proximal promoter region of the human RIIα gene may play important roles in modulating expression of the gene in human myometrial cells. We show that all three GC binding domains are involved in binding Sp1, Sp3, histone deacetylase (HDACs) 1/2 and RbAp48 transcriptional complexes. The functional significance of these binding domains was further analysed employing in vitro luciferase reporter assays with full-length/truncated RIIα promoter constructs. Importantly we show that treatment of primary human myometrial cell cultures with the general class I/II HDAC inhibitor trichostatin A results in an increase in mRNA/protein levels. Moreover the increase in mRNA levels appeared to be preceded by an increase in aH3, PolIIa, Sp3 and HDAC 2 binding to the three SpI-III (GC) binding sites within the RIIα promoter. These results enable us to provide a model whereby RIIα expression is epigenetically regulated in human myometrial smooth muscle cells by histone deacetylase(s) activity within the GC-rich proximal promoter region of the gene.

  2. Down-regulation of histone H2B by DNA-dependent protein kinase in response to DNA damage through modulation of octamer transcription factor 1.

    PubMed

    Schild-Poulter, Caroline; Shih, Amy; Yarymowich, Nicholas C; Haché, Robert J G

    2003-11-01

    Cells respond to double-stranded DNA breaks (DSBs) by pausing cell cycle progression to allow the repair machinery to restore genomic integrity. DNA-dependent protein kinase (DNA-PK), comprising a large catalytic subunit (DNA-PK(cs)) and the Ku antigen regulatory subunit (Ku70/Ku80), is activated in response to DSBs and is required for DNA repair through the nonhomologous end-joining pathway. Here we provide evidence that DNA-PK participates in altering specific gene expression in response to DNA damage by modulating the stability and transcriptional regulatory potential of the essential transcription factor octamer transcription factor 1 (Oct-1). Histone H2B and U2 RNA, whose expression are highly dependent on Oct-1, were strongly decreased in response to ionizing radiation in a DNA-PK-dependent manner, and Oct-1-dependent reporter gene transcription was repressed. Furthermore, Oct-1 phosphorylation in response to ionizing radiation increased in a DNA-PK-dependent manner. Paradoxically, down-regulation of transactivation correlated with the rapid DNA-PK-dependent stabilization of Oct-1. Stabilization of Oct-1 was dependent on the NH(2)-terminal region of Oct-1, which contains a transcriptional activation domain and which was phosphorylated by DNA-PK in vitro. These results suggest a mechanism for the regulation of Oct-1 in response to DNA damage through specific phosphorylation within the NH(2)-terminal transcriptional regulatory domain.

  3. Arabidopsis thaliana mitogen-activated protein kinase 6 is involved in seed formation and modulation of primary and lateral root development

    PubMed Central

    Guevara-García, A. A.

    2014-01-01

    Mitogen-activated protein kinase (MAPKs) cascades are signal transduction modules highly conserved in all eukaryotes regulating various aspects of plant biology, including stress responses and developmental programmes. In this study, we characterized the role of MAPK 6 (MPK6) in Arabidopsis embryo development and in post-embryonic root system architecture. We found that the mpk6 mutation caused altered embryo development giving rise to three seed phenotypes that, post-germination, correlated with alterations in root architecture. In the smaller seed class, mutant seedlings failed to develop the primary root, possibly as a result of an earlier defect in the division of the hypophysis cell during embryo development, but they had the capacity to develop adventitious roots to complete their life cycle. In the larger class, the MPK6 loss of function did not cause any evident alteration in seed morphology, but the embryo and the mature seed were bigger than the wild type. Seedlings developed from these bigger seeds were characterized by a primary root longer than that of the wild type, accompanied by significantly increased lateral root initiation and more and longer root hairs. Apparently, the increment in primary root growth resulted from an enhanced cell production and cell elongation. Our data demonstrated that MPK6 plays an important role during embryo development and acts as a repressor of primary and lateral root development. PMID:24218326

  4. Effects of modulators of AMP-activated protein kinase on TASK-1/3 and intracellular Ca2+ concentration in rat carotid body glomus cells

    PubMed Central

    Kim, Donghee; Kang1,2, Dawon; Martin, Elizabeth A.; Kim, Insook; Carroll, John L.

    2014-01-01

    Acute hypoxia depolarizes carotid body chemoreceptor (glomus) cells and elevates intracellular Ca2+ concentration ([Ca2+]i). Recent studies suggest that AMP-activated protein kinase (AMPK) mediates these effects of hypoxia by inhibiting the background K+ channels such as TASK. Here we studied the effects of modulators of AMPK on TASK activity in cell-attached patches. Activators of AMPK (1 mM AICAR and 0.1–0.5 mM A769662) did not inhibit TASK activity or cause depolarization during acute (10 min) or prolonged (2–3 hr) exposure. Hypoxia inhibited TASK activity by ~70% in cells pretreated with AICAR or A769662. Both AICAR and A769662 (15–40 min) failed to increase [Ca2+]i in glomus cells. Compound C (40 µM), an inhibitor of AMPK, showed no effect on hypoxia-induced inhibition of TASK. AICAR and A769662 phosphorylated AMPKα in PC12 cells, and Compound C blocked the phosphorylation. Our results suggest that AMPK does not affect TASK activity and is not involved in hypoxia-induced elevation of intracellular [Ca2+] in isolated rat carotid body glomus cells. PMID:24530802

  5. Hypertonicity regulates the function of human neutrophils by modulating chemoattractant receptor signaling and activating mitogen-activated protein kinase p38.

    PubMed Central

    Junger, W G; Hoyt, D B; Davis, R E; Herdon-Remelius, C; Namiki, S; Junger, H; Loomis, W; Altman, A

    1998-01-01

    Excessive neutrophil activation causes posttraumatic complications, which may be reduced with hypertonic saline (HS) resuscitation. We tested if this is because of modulated neutrophil function by HS. Clinically relevant hypertonicity (10-25 mM) suppressed degranulation and superoxide formation in response to fMLP and blocked the activation of the mitogen activated protein kinases (MAPK) ERK1/2 and p38, but did not affect Ca2+ mobilization. HS did not suppress oxidative burst in response to phorbol myristate acetate (PMA). This indicates that HS suppresses neutrophil function by intercepting signal pathways upstream of or apart from PKC. HS activated p38 by itself and enhanced degranulation in response to PKC activation. This enhancement was reduced by inhibition of p38 with SB203580, suggesting that p38 up-regulation participates in HS-induced enhancements of degranulation. HS had similar effects on the degranulation of cells that were previously stimulated with fMLP, but had no effect on its own, suggesting that HS enhancement of degranulation requires another signal. We conclude that depending on other stimuli, HS can suppress neutrophil activation by intercepting multiple receptor signals or augment degranulation by enhancing p38 signaling. In patients HS resuscitation may reduce posttraumatic complications by preventing neutrophil activation via chemotactic factors released during reperfusion. PMID:9637711

  6. Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases

    PubMed Central

    Clarke, Paul A; Ortiz-Ruiz, Maria-Jesus; TePoele, Robert; Adeniji-Popoola, Olajumoke; Box, Gary; Court, Will; Czasch, Stephanie; El Bawab, Samer; Esdar, Christina; Ewan, Ken; Gowan, Sharon; De Haven Brandon, Alexis; Hewitt, Phillip; Hobbs, Stephen M; Kaufmann, Wolfgang; Mallinger, Aurélie; Raynaud, Florence; Roe, Toby; Rohdich, Felix; Schiemann, Kai; Simon, Stephanie; Schneider, Richard; Valenti, Melanie; Weigt, Stefan; Blagg, Julian; Blaukat, Andree; Dale, Trevor C; Eccles, Suzanne A; Hecht, Stefan; Urbahns, Klaus; Workman, Paul; Wienke, Dirk

    2016-01-01

    Mediator-associated kinases CDK8/19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We discovered two series of potent chemical probes with high selectivity for CDK8/19. Despite pharmacodynamic evidence for robust on-target activity, the compounds exhibited modest, though significant, efficacy against human tumor lines and patient-derived xenografts. Altered gene expression was consistent with CDK8/19 inhibition, including profiles associated with super-enhancers, immune and inflammatory responses and stem cell function. In a mouse model expressing oncogenic beta-catenin, treatment shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. In two species, neither probe was tolerated at therapeutically-relevant exposures. The complex nature of the toxicity observed with two structurally-differentiated chemical series is consistent with on-target effects posing significant challenges to the clinical development of CDK8/19 inhibitors. DOI: http://dx.doi.org/10.7554/eLife.20722.001 PMID:27935476

  7. Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia*

    PubMed Central

    Roth Flach, Rachel J.; Danai, Laura V.; DiStefano, Marina T.; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B.; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K.; Bortell, Rita; Alonso, Laura C.; Czech, Michael P.

    2016-01-01

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo. After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. PMID:27226575

  8. Pathway illuminated: visualizing protein kinase C signaling.

    PubMed

    Violin, Jonathan D; Newton, Alexandra C

    2003-12-01

    Protein kinase C has been at the center of cell signaling since the discovery 25 years ago that it transduces signals that promote phospholipid hydrolysis. In recent years, the use of genetically encoded fluorescent reporters has enabled studies of the regulation of protein kinase C signaling in living cells. Advances in imaging techniques have unveiled unprecedented detail of the signal processing mechanics of protein kinase C, from the second messengers calcium and diacylglycerol that regulate protein kinase C activity, to the locations and kinetics of different protein kinase C isozymes, to the spatial and temporal dynamics of substrate phosphorylation by this key enzyme. This review discusses how fluorescence imaging studies have illuminated the fidelity with which protein kinase C transduces rapidly changing extracellular information into intracellular phosphorylation signals.

  9. ERK kinases modulate the activation of PI3 kinase related kinases (PIKKs) in DNA damage response.

    PubMed

    Lin, Xiaozeng; Yan, Judy; Tang, Damu

    2013-12-01

    DNA damage response (DDR) is the critical surveillance mechanism in maintaining genome integrity. The mechanism activates checkpoints to prevent cell cycle progression in the presence of DNA lesions, and mediates lesion repair. DDR is coordinated by three apical PI3 kinase related kinases (PIKKs), including ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-PKcs (the catalytic subunit of the DNA dependent protein kinase). These kinases are activated in response to specific DNA damage or lesions, resulting in checkpoint activation and DNA lesion repair. While it is clear that the pathways of ATM, ATR, and DNA-PK are the core components of DDR, there is accumulating evidence revealing the involvement of other cellular pathways in regulating DDR; this is in line with the concept that in addition to being a nuclear event DDR is also a cellular process. One of these pathways is the extracellular signal-regulated kinase (ERK) MAPK (mitogen-activated protein kinase) pathway. ERK is a converging point of multiple signal transduction pathways involved in cell proliferation, differentiation, and apoptosis. Adding to this list of pathways is the recent development of ERK in DDR. The ERK kinases (ERK1 and ERK2) contribute to the proper execution of DDR in terms of checkpoint activation and the repair of DNA lesions. This review summarizes the contributions of ERK to DDR with emphasis on the relationship of ERK kinases with the activation of ATM, ATR, and DNA-PKcs.

  10. Behavioral modulation of neuronal calcium/calmodulin-dependent protein kinase II activity: differential effects on nicotine-induced spinal and supraspinal antinociception in mice.

    PubMed

    Damaj, M Imad

    2007-10-15

    Recent studies have implicated the involvement of Ca(2+)-dependent mechanisms, in particular calcium/calmodulin-dependent protein kinase II (CaM kinase II) in nicotine-induced antinociception using the tail-flick test. The spinal cord was suggested as a possible site of this involvement. The present study was undertaken to investigate the hypothesis that similar mechanisms exist for nicotine-induced antinociception in the hot-plate test, a response thought to be centrally mediated. In order to assess these mechanisms, i.c.v. administered CaM kinase II inhibitors were evaluated for their effects on antinociception produced by either i.c.v. or s.c. administration of nicotine in both tests. In addition, nicotine's analgesic effects were tested in mice lacking half of their CaM kinase II (CaM kinase II heterozygous) and compare it to their wild-type counterparts. Our results showed that although structurally unrelated CaM kinase II inhibitors blocked nicotine's effects in the tail-flick test in a dose-related manner, they failed to block the hot-plate responses. In addition, the antinociceptive effects of systemic nicotine in the tail-flick but not the hot-plate test were significantly reduced in CaM kinase II heterozygous mice. These observations indicate that in contrast to the tail-flick response, the mechanism of nicotine-induced antinociception in the hot-plate test is not mediated primarily via CaM kinase II-dependent mechanisms at the supraspinal level.

  11. Pkh1 and Pkh2 Differentially Phosphorylate and Activate Ypk1 and Ykr2 and Define Protein Kinase Modules Required for Maintenance of Cell Wall Integrity

    PubMed Central

    Roelants, Françoise M.; Torrance, Pamela D.; Bezman, Natalie; Thorner, Jeremy

    2002-01-01

    Saccharomyces cerevisiae Pkh1 and Pkh2 are functionally redundant homologs of mammalian protein kinase, phosphoinositide-dependent protein kinase-1. They activate two closely related, functionally redundant enzymes, Ypk1 and Ykr2 (homologs of mammalian protein kinase, serum- and glucocorticoid-inducible protein kinase). We found that Ypk1 has a more prominent role than Ykr2 in mediating their shared essential function. Considerable evidence demonstrated that Pkh1 preferentially activates Ypk1, whereas Pkh2 preferentially activates Ykr2. Loss of Pkh1 (but not Pkh2) reduced Ypk1 activity; conversely, Pkh1 overexpression increased Ypk1 activity more than Pkh2 overexpression. Loss of Pkh2 reduced Ykr2 activity; correspondingly, Pkh2 overexpression increased Ykr2 activity more than Pkh1 overexpression. When overexpressed, a catalytically active C-terminal fragment (kinase domain) of Ypk1 was growth inhibitory; loss of Pkh1 (but not Pkh2) alleviated toxicity. Loss of Pkh2 (but not Pkh1) exacerbated the slow growth phenotype of a ypk1Δ strain. This Pkh1-Ypk1 and Pkh2-Ykr2 dichotomy is not absolute because all double mutants (pkh1Δ ypk1Δ, pkh2Δ ypk1Δ, pkh1Δ ykr2Δ, and pkh2Δ ykr2Δ) were viable. Compartmentation contributes to selectivity because Pkh1 and Ypk1 were located exclusively in the cytosol, whereas Pkh2 and Ykr2 entered the nucleus. At restrictive temperature, ypk1-1ts ykr2Δ cells lysed rapidly, but not in medium containing osmotic support. Dosage and extragenic suppressors were selected. Overexpression of Exg1 (major exoglucanase), or loss of Kex2 (endoprotease involved in Exg1 processing), rescued growth at high temperature. Viability was also maintained by PKC1 overexpression or an activated allele of the downstream protein kinase (BCK1-20). Conversely, absence of Mpk1 (distal mitogen-activated protein kinase of the PKC1 pathway) was lethal in ypk1-1ts ykr2Δ cells. Thus, Pkh1-Ypk1 and Pkh2-Ykr2 function in a novel pathway for cell wall integrity that

  12. A rice calcium-dependent protein kinase OsCPK12 oppositely modulates salt-stress tolerance and blast disease resistance.

    PubMed

    Asano, Takayuki; Hayashi, Nagao; Kobayashi, Michie; Aoki, Naohiro; Miyao, Akio; Mitsuhara, Ichiro; Ichikawa, Hiroaki; Komatsu, Setsuko; Hirochika, Hirohiko; Kikuchi, Shoshi; Ohsugi, Ryu

    2012-01-01

    Calcium-dependent protein kinases (CDPKs) regulate the downstream components in calcium signaling pathways. We investigated the effects of overexpression and disruption of an Oryza sativa (rice) CDPK (OsCPK12) on the plant's response to abiotic and biotic stresses. OsCPK12-overexpressing (OsCPK12-OX) plants exhibited increased tolerance to salt stress. The accumulation of hydrogen peroxide (H(2) O(2) ) in the leaves was less in OsCPK12-OX plants than in wild-type (WT) plants. Genes encoding reactive oxygen species (ROS) scavenging enzymes (OsAPx2 and OsAPx8) were more highly expressed in OsCPK12-OX plants than in WT plants, whereas the expression of the NADPH oxidase gene, OsrbohI, was decreased in OsCPK12-OX plants compared with WT plants. Conversely, a retrotransposon (Tos17) insertion mutant, oscpk12, and plants transformed with an OsCPK12 RNA interference (RNAi) construct were more sensitive to high salinity than were WT plants. The level of H(2) O(2) accumulation was greater in oscpk12 and OsCPK12 RNAi plants than in the WT. These results suggest that OsCPK12 promotes tolerance to salt stress by reducing the accumulation of ROS. We also observed that OsCPK12-OX seedlings had increased sensitivity to abscisic acid (ABA) and increased susceptibility to blast fungus, probably resulting from the repression of ROS production and/or the involvement of OsCPK12 in the ABA signaling pathway. Collectively, our results suggest that OsCPK12 functions in multiple signaling pathways, positively regulating salt tolerance and negatively modulating blast resistance.

  13. Multiple activation of mitogen-activated protein kinases by purified independent CCN2 modules in vascular endothelial cells and chondrocytes in culture.

    PubMed

    Kubota, S; Kawaki, H; Kondo, S; Yosimichi, G; Minato, M; Nishida, T; Hanagata, H; Miyauchi, A; Takigawa, M

    2006-12-01

    CCN2 consists of 4 distinct modules that are conserved among various CCN family protein members. From the N-terminus, insulin-like growth factor binding protein (IGFBP), von Willebrand factor type C repeat (VWC), thrombospondin type 1 repeat (TSP1) and C-terminal cysteine-knot (CT) modules are all aligned tandem therein. The multiple functionality of CCN2 is thought to be enabled by the differential use of these modules when interacting with other molecules. In this study, we independently prepared all 4 purified module proteins of human CCN2, utilizing a secretory production system with Brevibacillus choshinensis and thus evaluated the cell biological effects of such single modules. In human umbilical vascular endothelial cells (HUVECs), VWC, TSP and CT modules, as well as a full-length CCN2, were capable of efficiently activating the ERK signal transduction cascade, whereas IGFBP was not. In contrast, the IGFBP module was found to prominently activate JNK in human chondrocytic HCS-2/8 cells, while the others showed similar effects at lower levels. In addition, ERK1/2 was modestly, but significantly activated by IGFBP and VWC in those cells. No single module, but a mixture of the 4 modules provoked a significant activation of p38 MAPK in HCS-2/8 cells, which was activated by the full-length CCN2. Therefore, the signals emitted by CCN2 can be highly differential, depending upon the cell types, which are thus enabled by the tetramodular structure. Furthermore, the cell biological effects of each module on these cells were also evaluated to clarify the relationship among the modules, the signaling pathways and biological outcomes. Our present results not only demonstrate that single CCN2 modules were potent activators of the intracellular signaling cascade to yield a biological response per se, while also providing new insight into the module-wise structural and functional relationship of a prototypic CCN family member, CCN2.

  14. Phosphorylation of Krüppel-like Factor 3 (KLF3/BKLF) and C-terminal Binding Protein 2 (CtBP2) by Homeodomain-interacting Protein Kinase 2 (HIPK2) Modulates KLF3 DNA Binding and Activity*

    PubMed Central

    Dewi, Vitri; Kwok, Alister; Lee, Stella; Lee, Ming Min; Tan, Yee Mun; Nicholas, Hannah R.; Isono, Kyo-ichi; Wienert, Beeke; Mak, Ka Sin; Knights, Alexander J.; Quinlan, Kate G. R.; Cordwell, Stuart J.; Funnell, Alister P. W.; Pearson, Richard C. M; Crossley, Merlin

    2015-01-01

    Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3. PMID:25659434

  15. Measuring protein kinase and sugar kinase activity in plant pathogenic fusarium species.

    PubMed

    Bluhm, Burton H; Zhao, Xinhua

    2010-01-01

    As ubiquitous metabolic and signaling intermediaries, kinases regulate innumerable aspects of fungal growth and development. At its simplest, the enzymatic function of a kinase is to transfer a phosphate from a donor molecule (such as adenosine triphosphate) to an acceptor molecule, such as a protein, carbohydrate, or lipid. Kinase activity is intricately interwoven into signal transduction, and ultimately modulates gene expression, downstream phosphorylation events, and other mechanisms of posttranslational modification. Therefore, sensitive and reproducible techniques to measure kinase activity are crucial to elucidate cellular signaling and for fungal functional genomics.Protein and sugar kinases regulate multiple aspects of pathogenesis in the mycotoxigenic, plant pathogenic fungi Fusarium graminearum, and Fusarium verticillioides. Here, we present protocols to (1) quantify phosphorylation of mitogen-activated protein kinases in F. graminearum, and (2) determine glucokinase activity in F. verticillioides. The mitogen-activated protein kinase phosphorylation assay utilizes immunological methods to quantify substrate phosphorylation, whereas the glucokinase assay is a coupled enzyme assay, in which phosphorylation of glucose by glucokinase is measured indirectly through the subsequent reduction of NADP+ to NADPH, a substrate more amenable for spectrophotometric detection.

  16. The Predikin webserver: improved prediction of protein kinase peptide specificity using structural information

    PubMed Central

    Saunders, Neil F. W.

    2008-01-01

    The Predikin webserver allows users to predict substrates of protein kinases. The Predikin system is built from three components: a database of protein kinase substrates that links phosphorylation sites with specific protein kinase sequences; a perl module to analyse query protein kinases and a web interface through which users can submit protein kinases for analysis. The Predikin perl module provides methods to (i) locate protein kinase catalytic domains in a sequence, (ii) classify them by type or family, (iii) identify substrate-determining residues, (iv) generate weighted scoring matrices using three different methods, (v) extract putative phosphorylation sites in query substrate sequences and (vi) score phosphorylation sites for a given kinase, using optional filters. The web interface provides user-friendly access to each of these functions and allows users to obtain rapidly a set of predictions that they can export for further analysis. The server is available at http://predikin.biosci.uq.edu.au. PMID:18477637

  17. Cyclic-GMP-dependent protein kinase inhibits the Ras/Mitogen-activated protein kinase pathway.

    PubMed

    Suhasini, M; Li, H; Lohmann, S M; Boss, G R; Pilz, R B

    1998-12-01

    Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3', 5'-cyclic monophosphate and 8-bromoguanosine-3',5'-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Ibeta expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser43, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser43 uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser43 was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser43 in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser43 phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by

  18. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2001-07-03

    The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  19. Molecular cloning of plant transcripts encoding protein kinase homologs.

    PubMed Central

    Lawton, M A; Yamamoto, R T; Hanks, S K; Lamb, C J

    1989-01-01

    Oligonucleotides, corresponding to conserved regions of animal protein-serine/threonine kinases, were used to isolate cDNAs encoding plant homologs in the dicot bean (Phaseolus vulgaris L.) and the monocot rice (Oryzae sativa L.). The C-terminal regions of the deduced polypeptides encoded by the bean (PVPK-1) and rice (G11A) cDNAs, prepared from mRNAs of suspension cultures and leaves, respectively, contain features characteristic of the catalytic domains of eukaryotic protein-serine/threonine kinases, indicating that these cDNAs encode plant protein kinases. The putative catalytic domains are most closely related to cyclic nucleotide-dependent protein kinases and the protein kinase C family, suggesting the plant homologs may likewise transduce extracellular signals. However, outside these domains, PVPK-1 and G11A exhibit no homology either to each other or to regulatory domains of other protein kinases, indicating the plant homologs are modulated by other signals. PVPK-1 corresponds to a 2.4-kb transcript in suspension cultured bean cells. Southern blots of genomic DNA indicate that PVPK-1 and G11A correspond to single copy genes that form part of a family of related plant sequences. Images PMID:2541432

  20. The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinases (MAPKAPKs) in Inflammation

    PubMed Central

    Moens, Ugo; Kostenko, Sergiy; Sveinbjørnsson, Baldur

    2013-01-01

    Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed. PMID:24705157

  1. Role of Protein Kinase C, PI3-kinase and Tyrosine Kinase in Activation of MAP Kinase by Glucose and Agonists of G-protein Coupled Receptors in INS-1 Cells

    PubMed Central

    Böcker, Dietmar

    2001-01-01

    MAP (mitogen-activated protein) kinase (also called Erk 1/2) plays a crucial role in cell proliferation and differentiation. Its impact on secretory events is less well established. The interplay of protein kinase C (PKC), PI3-kinase nd cellular tyrosine kinase with MAP kinase activity using inhibitors and compounds such as glucose, phorbol 12-myristate 13-acetate (PMA) and agonists of G-protein coupled receptors like gastrin releasing peptide (GRP), oxytocin (OT) and glucose-dependent insulinotropic peptide (GIP) was investigated in INS-1 cells, an insulin secreting cell line. MAP kinase activity was determined by using a peptide derived from the EGF receptor as a MAP kinase substrate and [ P 32 ]ATP. Glucose as well as GRP, OT and GIP exhibited a time-dependent increase in MAP kinase activity with a maximum at time point 2.5 min. All further experiments were performed using 2.5 min incubations. The flavone PD 098059 is known to bind to the inactive forms of MEK1 (MAPK/ERK-Kinase) thus preventing activation by upstream activators. 20 μM PD 098059 ( IC 50 =51 μM) inhibited MAP kinase stimulated by either glucose, GRP, OT, GIP or PMA. Inhibiton (“downregulation”) of PKC by a long term (22h) pretreatment with 1 μM PMA did not influence MAP kinase activity when augmented by either of the above mentioned compound. To investigate whether PI3-kinase and cellular tyrosine kinase are involved in G-protein mediated effects on MAP kinase, inhibitors were used: 100 nM wortmannin (PI3-kinase inhibitor) reduced the effects of GRP, OT and GIP but not that of PMA; 100 μM genistein (tyrosine kinase inhibitor) inhibited the stimulatory effect of either above mentioned compound on MAP kinase activation. Inhibition of MAP kinase by 20 μM PD 098059 did not influence insulin secretion modulated by either compound (glucose, GRP, OT or GIP). [ H 3 ]Thymidine incorporation, however, was severely inhibited by PD 098059. Thus MAP kinase is important for INS-1 cell proliferation but

  2. Blockade of ankyrin repeat-rich membrane spanning protein modulates extracellular signal-regulated kinase expression and inhibits allergic inflammation in ovalbumin-sensitized mice.

    PubMed

    Ni, Xiuqin; Li, Xing; Tao, Shuhua; Xu, Minghui; Ma, Hongmei; Wang, Xiuli

    2013-07-01

    Ankyrin repeat-rich membrane spanning protein (ARMS), also known as kinase D-interacting substrate of 220 kDa (Kidins220), is a transmembrane protein that has been reported to be involved in the pathogenesis of asthma through the nerve growth factor (NGF)/tyrosine kinase A (TrkA) receptor signaling pathway. To investigate whether NGF/TrkA-Kidins220/ARMS-extracellular signal-regulated kinase (ERK) signaling is activated in airway inflammation of asthma, BALB/c mice were sensitized and challenged with ovalbumin (OVA). The effects of Kidins220/ARMS on ERK, interleukin (IL)-1β, IL-4 and tumor necrosis factor (TNF)-α in lung tissues following the allergic airway challenge in mice were assessed by administering anti-ARMS antibody to the mice. Pathological changes in the bronchi and lung tissues were examined via hematoxylin and eosin staining. The phosphorylated ERK, IL-1β, IL-4 and TNF-α levels were determined using western blot analysis and ELISA and were found to be overexpressed in lung tissues following the allergen challenge. Moreover, after the mice were treated with anti-NGF, anti-TrkA or anti-ARMS, the levels of Kidins220/ARMS, phosphorylated ERK, IL-1β, IL-4, TNF-α and allergen-induced airway inflammation were downregulated. These results suggested that NGF/TrkA-Kidins220/ARMS-ERK signaling was activated in airway inflammation induced by the allergic airway challenge, possibly representing a new mechanism in asthma.

  3. Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases*

    PubMed Central

    Lai, Shenshen; Safaei, Javad

    2016-01-01

    The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies. PMID:26742849

  4. Targeting AMP-activated protein kinase in adipocytes to modulate obesity-related adipokine production associated with insulin resistance and breast cancer cell proliferation

    PubMed Central

    2011-01-01

    Background Adipokines, e.g. TNFα, IL-6 and leptin increase insulin resistance, and consequent hyperinsulinaemia influences breast cancer progression. Beside its mitogenic effects, insulin may influence adipokine production from adipocyte stromal cells and paracrine enhancement of breast cancer cell growth. In contrast, adiponectin, another adipokine is protective against breast cancer cell proliferation and insulin resistance. AMP-activated protein kinase (AMPK) activity has been found decreased in visceral adipose tissue of insulin-resistant patients. Lipopolysaccharides (LPS) link systemic inflammation to high fat diet-induced insulin resistance. Modulation of LPS-induced adipokine production by metformin and AMPK activation might represent an alternative way to treat both, insulin resistance and breast cancer. Methods Human preadipocytes obtained from surgical biopsies were expanded and differentiated in vitro into adipocytes, and incubated with siRNA targeting AMPKalpha1 (72 h), LPS (24 h, 100 μg/ml) and/or metformin (24 h, 1 mM) followed by mRNA extraction and analyses. Additionally, the supernatant of preadipocytes or derived-adipocytes in culture for 24 h was used as conditioned media to evaluate MCF-7 breast cancer cell proliferation. Results Conditioned media from preadipocyte-derived adipocytes, but not from undifferentiated preadipocytes, increased MCF-7 cell proliferation (p < 0.01). Induction of IL-6 mRNA by LPS was reduced by metformin (p < 0.01), while the LPS-induced mRNA expression of the naturally occurring anti-inflammatory cytokine interleukin 1 receptor antagonist was increased (p < 0.01). Silencing of AMPKalpha1 enhanced LPS-induced IL-6 and IL-8 mRNA expression (p < 0.05). Conclusions Adipocyte-secreted factors enhance breast cancer cell proliferation, while AMPK and metformin improve the LPS-induced adipokine imbalance. Possibly, AMPK activation may provide a new way not only to improve the obesity-related adipokine profile and insulin

  5. Troponin I Mutations R146G and R21C Alter Cardiac Troponin Function, Contractile Properties, and Modulation by Protein Kinase A (PKA)-mediated Phosphorylation*

    PubMed Central

    Cheng, Yuanhua; Rao, Vijay; Tu, An-yue; Lindert, Steffen; Wang, Dan; Oxenford, Lucas; McCulloch, Andrew D.; McCammon, J. Andrew; Regnier, Michael

    2015-01-01

    Two hypertrophic cardiomyopathy-associated cardiac troponin I (cTnI) mutations, R146G and R21C, are located in different regions of cTnI, the inhibitory peptide and the cardiac-specific N terminus. We recently reported that these regions may interact when Ser-23/Ser-24 are phosphorylated, weakening the interaction of cTnI with cardiac TnC. Little is known about how these mutations influence the affinity of cardiac TnC for cTnI (KC-I) or contractile kinetics during β-adrenergic stimulation. Here, we tested how cTnIR146G or cTnIR21C influences contractile activation and relaxation and their response to protein kinase A (PKA). Both mutations significantly increased Ca2+ binding affinity to cTn (KCa) and KC-I. PKA phosphorylation resulted in a similar reduction of KCa for all complexes, but KC-I was reduced only with cTnIWT. cTnIWT, cTnIR146G, and cTnIR21C were complexed into cardiac troponin and exchanged into rat ventricular myofibrils, and contraction/relaxation kinetics were measured ± PKA phosphorylation. Maximal tension (Tmax) was maintained for cTnIR146G- and cTnIR21C-exchanged myofibrils, and Ca2+ sensitivity of tension (pCa50) was increased. PKA phosphorylation decreased pCa50 for cTnIWT-exchanged myofibrils but not for either mutation. PKA phosphorylation accelerated the early slow phase relaxation for cTnIWT myofibrils, especially at Ca2+ levels that the heart operates in vivo. Importantly, this effect was blunted for cTnIR146G- and cTnIR21C-exchanged myofibrils. Molecular dynamics simulations suggest both mutations inhibit formation of intra-subunit contacts between the N terminus and the inhibitory peptide of cTnI that is normally seen with WT-cTn upon PKA phosphorylation. Together, our results suggest that cTnIR146G and cTnIR21C blunt PKA modulation of activation and relaxation kinetics by prohibiting cardiac-specific N-terminal interaction with the cTnI inhibitory peptide. PMID:26391394

  6. Fullerene (C60) nanoparticles exert photocytotoxicity through modulation of reactive oxygen species and p38 mitogen-activated protein kinase activation in the MCF-7 cancer cell line

    NASA Astrophysics Data System (ADS)

    Li, Zhi; Zhang, Fei-long; Wang, Zhiyuan; Pan, Li-li; Shen, Ying-ying; Zhang, Zhen-zhong

    2013-12-01

    The photocytotoxicity of water-dispersed 100-300 nm fullerene amino acid derivatives nanoparticles was studied. The nanoparticle solution of fullerene derivatives, l-phenylalanine (C60-phe) and glycine (C60-gly), suppressed the in vitro growth of MCF-7 cells lines, induced cancer cells apoptosis, and caused a perturbation of the cell cycle. These nanoparticle solutions increased intracellular reactive oxygen species after irradiation. C60-phe or C60-gly upregulated the expression of phosphorylated (p)p38 mitogen-activated protein kinase (MAPK). N-Acetyl- l-cysteine significantly depressed the composite-induced activation of p38MAPK, and the kinase inhibitor SB203580 significantly prevented C60 derivative-induced cell apoptosis. This study revealed that p38MAPK is activated by C60 nanoparticles through triggering reactive oxygen species generation, leading to cancer cell injuries.

  7. Homeodomain-interacting Protein Kinase-2 (HIPK2) Phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77 and Modulates Its DNA Binding Affinity

    PubMed Central

    Zhang, Qingchun; Wang, Yinsheng

    2008-01-01

    The chromosomal high-mobility group A (HMGA) proteins, comprising of HMGA1a, HMGA1b and HMGA2, play important roles in the regulation of numerous processes in eukaryotic cells, such as transcriptional regulation, DNA repair, RNA processing, and chromatin remodeling. The biological activities of HMGA1 proteins are highly regulated by their post-translational modifications (PTMs), including acetylation, methylation and phosphorylation. Recently, it was found that the homeodomain-interacting protein kinase-2 (HIPK2), a newly identified serine/threonine kinase, co-immunoprecipitated with, and phosphorylated HMGA1 proteins. However, the sites and the biological significance of the phosphorylation have not been elucidated. Here, we found that HIPK2 phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77, and HMGA1b at Thr-41 and Thr-66. In addition, we demonstrated that cdc2, which is known to phosphorylate HMGA1 proteins, could induce the phosphorylation of HMGA1 proteins at the same Ser/Thr sites. The two kinases, however, exhibited different site preferences for the phosphorylation: The preference for HIPK2 phosphorylation followed the order of Thr-77 > Thr-52 > Ser-35, whereas the order for cdc2 phosphorylation was Thr-52 > Thr-77 > Ser-35. Moreover, we found that the HIPK2-phosphorylated HMGA1a reduced the binding affinity of HMGA1a to human germ line ε promoter, and the drop in binding affinity induced by HIPK2 phosphorylation was lower than that introduced by cdc2 phosphorylation, which is consistent with the notion that the second AT-hook in HMGA1a is more important for DNA binding than the third AT-hook. Synopsis Here we report that both HIPK2 and cdc2 phosphorylate HMGA1a at Ser-35, Thr-52 and Thr-77, but the two kinases exhibit different site preferences. Moreover, we found that HIPK2-induced phosphorylation of HMGA1a reduced the binding affinity of HMGA1a to DNA, and the drop in binding affinity was lower than that introduced by cdc2 phosphorylation, confirming

  8. Cadmium activates a mitogen-activated protein kinase gene and MBP kinases in rice.

    PubMed

    Yeh, Chuan-Ming; Hsiao, Lin-June; Huang, Hao-Jen

    2004-09-01

    Mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. In plants, it has been evidenced that MAPKs play a role in the signaling of biotic and abiotic stresses, plant hormones, and cell cycle cues. However, the effect of heavy metals on plant MAPKs has not been well examined. The Northern blot analysis of OsMAPK mRNA levels has shown that only OsMAPK2, but not OsMAPK3 and OsMAPK4, expressed in suspension-cultured cells in response to 100-400 microM Cd treatments. The OsMAPK2 transcripts increased within 12 h upon 400 microM Cd treatment. In addition, we found that 42- and 50-kDa MBP kinases were significantly activated by Cd treatment in rice suspension-cultured cells. And 40-, 42-, 50- and 64-kDa MBP kinases were activated in rice roots. Furthermore, GSH inhibits Cd-induced 40-kDa MBP kinase activation. By immunoblot analysis and immunoprecipitation followed by in-gel kinase assay, we confirmed that Cd-activated 42-kDa MBP kinase is a MAP kinase. Our results suggest that a MAP kinase cascade may function in the Cd-signalling pathway in rice.

  9. Apoptosis of HL-60 human leukemia cells induced by Asiatic acid through modulation of B-cell lymphoma 2 family proteins and the mitogen-activated protein kinase signaling pathway.

    PubMed

    Wu, Qiuling; Lv, Tingting; Chen, Yan; Wen, Lu; Zhang, Junli; Jiang, Xudong; Liu, Fang

    2015-07-01

    The toxicities of conventional chemotherapeutic agents to normal cells restrict their dosage and clinical efficacy in acute leukemia; therefore, it is important to develop novel chemotherapeutics, including natural products, which selectively target cancer-specific pathways. The present study aimed to explore the effect of the chemopreventive agent asiatic acid (AA) on the proliferation and apoptotic rate of the leukemia cell line HL-60 and investigated the mechanisms underlying its anti-tumor activity. The effect of AA on the proliferation of HL-60 cells was evaluated using the MTT assay. Annexin V-fluorescein isothiocyanate/propidium iodide double staining followed by flow cytometric analysis as well as Hoechst 33258 staining were used to analyze the apoptotic rate of the cells. Furthermore, changes of survivin, B-cell lymphoma 2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 expressions were detected by western blot analysis. AA blocked the growth of HL-60 cells in a dose- and time-dependent manner. The IC50-value of AA on HL-60 cells was 46.67 ± 5.08 µmol/l for 24 h. AA induced apoptosis in a dose-dependent manner, which was inhibited in the presence of Z-DEVD-FMK, a specific inhibitor of caspase. The anti-apoptotic proteins Bcl-2, Mcl-1 and survivin were downregulated by AA in a dose-dependent manner. Concurrently, AA inhibited ERK and p38 phosphorylation in a dose-dependent manner, while JNK phosphorylation was not affected. In conclusion, the present study indicated that the p38 and ERK pathways, as well as modulation of Bcl-2 family and survivin proteins were key regulators of apoptosis induced in HL-60 cells in response to AA.

  10. Regulation of death induction and chemosensitizing action of 3-bromopyruvate in myeloid leukemia cells: energy depletion, oxidative stress, and protein kinase activity modulation.

    PubMed

    Calviño, Eva; Estañ, María Cristina; Sánchez-Martín, Carlos; Brea, Rocío; de Blas, Elena; Boyano-Adánez, María del Carmen; Rial, Eduardo; Aller, Patricio

    2014-02-01

    3-Bromopyruvate (3-BrP) is an alkylating, energy-depleting drug that is of interest in antitumor therapies, although the mechanisms underlying its cytotoxicity are ill-defined. We show here that 3-BrP causes concentration-dependent cell death of HL60 and other human myeloid leukemia cells, inducing both apoptosis and necrosis at 20-30 μM and a pure necrotic response at 60 μM. Low concentrations of 3-BrP (10-20 μM) brought about a rapid inhibition of glycolysis, which at higher concentrations was followed by the inhibition of mitochondrial respiration. The combination of these effects causes concentration-dependent ATP depletion, although this cannot explain the lethality at intermediate 3-BrP concentrations (20-30 μM). The oxidative stress caused by exposure to 3-BrP was evident as a moderate overproduction of reactive oxygen species and a concentration-dependent depletion of glutathione, which was an important determinant of 3-BrP toxicity. In addition, 3-BrP caused glutathione-dependent stimulation of p38 mitogen-activated protein kinase (MAPK), mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK), and protein kinase B (Akt)/mammalian target of rapamycin/p70S6K phosphorylation or activation, as well as rapid LKB-1/AMP kinase (AMPK) activation, which was later followed by Akt-mediated inactivation. Experiments with pharmacological inhibitors revealed that p38 MAPK activation enhances 3-BrP toxicity, which is conversely restrained by ERK and Akt activity. Finally, 3-BrP was seen to cooperate with antitumor agents like arsenic trioxide and curcumin in causing cell death, a response apparently mediated by both the generation of oxidative stress induced by 3-BrP and the attenuation of Akt and ERK activation by curcumin. In summary, 3-BrP cytotoxicity is the result of several combined regulatory mechanisms that might represent important targets to improve therapeutic efficacy.

  11. Involvement of protein kinase C in the modulation of morphine-induced analgesia and the inhibitory effects of exposure to 60-hz magnetic fields in the land snail, Cepaea nemoralis

    SciTech Connect

    Kavaliers, M.; Ossenkopp, K.P. )

    1990-02-26

    One of the more consistent and dramatic effects of exposure to magnetic fields is the attenuation of morphine-induced analgesia. Results of previous studies have implicated alterations in calcium channel functioning and Ca{sup ++} flux in the mediation of these effects. It is generally accepted that Ca{sup ++}-activated-phospholipid-dependent protein kinase (Protein kinase C; PKC) plays an important role in relaying trans-membrane signaling in diverse Ca{sup ++} dependent cellular processes. In experiment 1 we observed that morphine-induced analgesia in the land snail, Cepaea nemoralis, as measured by the latency of an avoidance behavior to a warmed surface, was reduced by the PKC activator, SC-9, and was enhanced by the PKC inhibitors, H-7 and H-9. In contrast, HA-10004, a potent inhibitor of other protein kinases, but only a very weak inhibitor of PKC, had no effect on morphine-induced analgesia. In experiment 2 exposure of snails for 30 minutes to a 1.0 gauss (rms) 60-Hz magnetic field reduced morphine-induced analgesia. This inhibitory effect of the magnetic field was reduced by the PKC inhibitors, H-7 and H-9, and was augmented by the PKC activator SC-9. These results suggest that: (i) PKC is involved in the modulation of morphine-induced analgesia and, (ii) the inhibitory effects of magnetic fields involve PKC.

  12. TRAF7 protein promotes Lys-29-linked polyubiquitination of IkappaB kinase (IKKgamma)/NF-kappaB essential modulator (NEMO) and p65/RelA protein and represses NF-kappaB activation.

    PubMed

    Zotti, Tiziana; Uva, Antonio; Ferravante, Angela; Vessichelli, Mariangela; Scudiero, Ivan; Ceccarelli, Michele; Vito, Pasquale; Stilo, Romania

    2011-07-01

    Tumor necrosis factor receptor-associated factor (TRAF) proteins are cytoplasmic regulatory molecules that function as signal transducers for receptors involved in both innate and adaptive humoral immune responses. In this study, we show that TRAF7, the unique noncanonical member of the TRAF family, physically associates with IκB kinase/NF-κB essential modulator (NEMO) and with the RelA/p65 (p65) member of the NF-κB transcription factor family. TRAF7 promotes Lys-29-linked polyubiquitination of NEMO and p65 that results in lysosomal degradation of both proteins and altered activation. TRAF7 also influences p65 nuclear distribution. Microarray expression data are consistent with an inhibitory role for TRAF7 on NF-κB and a positive control of AP-1 transcription factor. Finally, functional data indicate that TRAF7 promotes cell death. Thus, this study identifies TRAF7 as a NEMO- and p65-interacting molecule and brings important information on the ubiquitination events that control NF-κB transcriptional activity.

  13. TRAF7 Protein Promotes Lys-29-linked Polyubiquitination of IκB Kinase (IKKγ)/NF-κB Essential Modulator (NEMO) and p65/RelA Protein and Represses NF-κB Activation

    PubMed Central

    Zotti, Tiziana; Uva, Antonio; Ferravante, Angela; Vessichelli, Mariangela; Scudiero, Ivan; Ceccarelli, Michele; Vito, Pasquale; Stilo, Romania

    2011-01-01

    Tumor necrosis factor receptor-associated factor (TRAF) proteins are cytoplasmic regulatory molecules that function as signal transducers for receptors involved in both innate and adaptive humoral immune responses. In this study, we show that TRAF7, the unique noncanonical member of the TRAF family, physically associates with IκB kinase/NF-κB essential modulator (NEMO) and with the RelA/p65 (p65) member of the NF-κB transcription factor family. TRAF7 promotes Lys-29-linked polyubiquitination of NEMO and p65 that results in lysosomal degradation of both proteins and altered activation. TRAF7 also influences p65 nuclear distribution. Microarray expression data are consistent with an inhibitory role for TRAF7 on NF-κB and a positive control of AP-1 transcription factor. Finally, functional data indicate that TRAF7 promotes cell death. Thus, this study identifies TRAF7 as a NEMO- and p65-interacting molecule and brings important information on the ubiquitination events that control NF-κB transcriptional activity. PMID:21518757

  14. Intracerebral administration of protein kinase A or cAMP response element-binding protein antisense oligonucleotide can modulate amphetamine-mediated appetite suppression in free-moving rats.

    PubMed

    Hsieh, Yih-Shou; Yang, Shun-Fa; Kuo, Dong-Yih

    2007-01-01

    Although amphetamine (AMPH)-induced appetite suppression has been attributed to its inhibitory action on neuropeptide Y (NPY), an appetite neurotransmitter abundant in the brain, molecular mechanisms underlying this effect are not well known. This study examined the possible role of protein kinase A (PKA) and cAMP response element-binding protein (CREB) signaling in this anorectic effect, and the results showed that both PKA and CREB mRNA levels in hypothalamus were increased following AMPH treatment, which was relevant to a reduction of NPY mRNA level. To determine whether PKA or CREB was involved in the anorectic response, intracerebroventricular infusions of antisense oligonucleotide (or missense control) were performed 60 min before daily AMPH treatment in conscious rats, and results showed that either PKA or CREB knockdown could block AMPH-induced anorexia as well as restore NPY mRNA level, indicating the respective involvement of PKA and CREB signaling in the regulation of NPY gene expression. It is suggested that hypothalamic PKA and CREB signaling may involve the central regulation of AMPH-mediated feeding suppression via the modulation of NPY gene expression.

  15. Blockade of ankyrin repeat-rich membrane spanning protein modulates extracellular signal-regulated kinase expression and inhibits allergic inflammation in ovalbumin-sensitized mice

    PubMed Central

    NI, XIUQIN; LI, XING; TAO, SHUHUA; XU, MINGHUI; MA, HONGMEI; WANG, XIULI

    2013-01-01

    Ankyrin repeat-rich membrane spanning protein (ARMS), also known as kinase D-interacting substrate of 220 kDa (Kidins220), is a transmembrane protein that has been reported to be involved in the pathogenesis of asthma through the nerve growth factor (NGF)/tyrosine kinase A (TrkA) receptor signaling pathway. To investigate whether NGF/TrkA-Kidins220/ARMS-extracellular signal-regulated kinase (ERK) signaling is activated in airway inflammation of asthma, BALB/c mice were sensitized and challenged with ovalbumin (OVA). The effects of Kidins220/ARMS on ERK, interleukin (IL)-1β, IL-4 and tumor necrosis factor (TNF)-α in lung tissues following the allergic airway challenge in mice were assessed by administering anti-ARMS antibody to the mice. Pathological changes in the bronchi and lung tissues were examined via hematoxylin and eosin staining. The phosphorylated ERK, IL-1β, IL-4 and TNF-α levels were determined using western blot analysis and ELISA and were found to be overexpressed in lung tissues following the allergen challenge. Moreover, after the mice were treated with anti-NGF, anti-TrkA or anti-ARMS, the levels of Kidins220/ARMS, phosphorylated ERK, IL-1β, IL-4, TNF-α and allergen-induced airway inflammation were downregulated. These results suggested that NGF/TrkA-Kidins220/ARMS-ERK signaling was activated in airway inflammation induced by the allergic airway challenge, possibly representing a new mechanism in asthma. PMID:24649008

  16. Phosphorylation of actin-binding protein (ABP-280; filamin) by tyrosine kinase p56lck modulates actin filament cross-linking.

    PubMed

    Pal Sharma, C; Goldmann, Wolfgang H

    2004-01-01

    Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, we decided to investigate the possibility of whether it serves as substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins. The interaction of p56lck with membrane glycoproteins is important for cell development and functional activation. Here, we show that purified p56lck interacts and catalyzes in vitro kinase reactions. Tyrosine phosphorylation by p56lck is restricted to a single peptide of labeled ABP-280 shown by protease digest. The addition of phorbol ester to cells results in the inhibition of phosphorylation of ABP-280 by p56lck. These results show a decrease in phosphorylation suggesting conformationally induced regulation. Dynamic light scattering confirmed increased actin filament cross-linking due to phosphorylation of ABP-280 by p56lck.

  17. Ca/sup 2 +/-stimulated catecholamine release from. cap alpha. -toxin-permeabilized PC12 cells: biochemical evidence for exocytosis and its modulation by protein kinase C and G protein

    SciTech Connect

    Ahnert-Hilger, G.; Braeutigam, M.; Gratzl, M.

    1987-12-01

    Two possible cellular pathways of catecholamines from the chromaffin vesicles of PC12 cells to the surrounding medium are explored in this study. The direct one circumventing the cytoplasm can be activated in ..cap alpha..-toxin-permeabilized cells with micromolar levels of free Ca/sup 2 +/. Catecholamine metabolites formed in the cytoplasm (i.e., 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylethanol) are neither formed nor released from the cells under these conditions. However, when vesicular catecholamines were discharged into the cytoplasm by addition of the ionophore nigericin, such metabolites are formed and released into the medium independent of Ca/sup 2 +/. Both types of experiments provide direct evidence for the operation of Ca/sup 2 +/-induced exocytosis of dopamine and noradrenaline in permeabilized PC12 cells. The Ca/sup 2 +/ dependence of dopamine or noradrenaline release, as measured by the determination of the endogenous catecholamines using the high-performance liquid chromatography technique, exhibits two different phases. One is already activated below ..mu..M free Ca/sup 2 +/ and plateaus at 1-5 ..mu..M free Ca/sup 2 +/, while a second occurs in the presence of larger amounts of free Ca/sup 2 +/ (10-100 ..mu..M). Ca/sup 2 +/-induced catecholamine release from the permeabilized cells can be modulated in different ways: It is enhanced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate and the diacylglycerol 1-oleyl-2-acetylglycerol provided Mg/sup 2 +//ATP is present, and it is inhibited by guanosine 5'-O-(3-thiotriphosphate). The latter effect is abolished by pretreatment of the cells with pertussis toxin but not by cholera toxin. Thus, it appears that Ca/sup 2 +/-induced exocytosis can be modulated via the protein kinase C system, as well as via GTP binding proteins.

  18. Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells.

    PubMed

    Sipos, Adrienn; Iván, Judit; Bécsi, Bálint; Darula, Zsuzsanna; Tamás, István; Horváth, Dániel; Medzihradszky, Katalin F; Erdődi, Ferenc; Lontay, Beáta

    2017-01-11

    Myosin phosphatase (MP) holoenzyme is a protein phosphatase-1 (PP1) type Ser/Thr specific enzyme that consists of a PP1 catalytic (PP1c) and a myosin phosphatase target subunit-1 (MYPT1). MYPT1 is an ubiquitously expressed isoform and it targets PP1c to its substrates. We identified the protein arginine methyltransferase 5 (PRMT5) enzyme of the methylosome complex as a MYPT1-binding protein uncovering the nuclear MYPT1-interactome of hepatocellular carcinoma cells. It is shown that PRMT5 is regulated by phosphorylation at Thr80 by RhoA-associated protein kinase and MP. Silencing of MYPT1 increased the level of the PRMT5-specific symmetric dimethylation on arginine residues of histone 2 A/4, a repressing gene expression mark, and it resulted in a global change in the expression of genes affecting cellular processes like growth, proliferation and cell death, also affecting the expression of the retinoblastoma protein and c-Myc. The phosphorylation of the MP inhibitory MYPT1(T850) and the regulatory PRMT5(T80) residues as well as the symmetric dimethylation of H2A/4 were elevated in human hepatocellular carcinoma and in other types of cancers. These changes correlated positively with the grade and state of the tumors. Our results suggest the tumor suppressor role of MP via inhibition of PRMT5 thereby regulating gene expression through histone arginine dimethylation.

  19. Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells

    PubMed Central

    Sipos, Adrienn; Iván, Judit; Bécsi, Bálint; Darula, Zsuzsanna; Tamás, István; Horváth, Dániel; Medzihradszky, Katalin F.; Erdődi, Ferenc; Lontay, Beáta

    2017-01-01

    Myosin phosphatase (MP) holoenzyme is a protein phosphatase-1 (PP1) type Ser/Thr specific enzyme that consists of a PP1 catalytic (PP1c) and a myosin phosphatase target subunit-1 (MYPT1). MYPT1 is an ubiquitously expressed isoform and it targets PP1c to its substrates. We identified the protein arginine methyltransferase 5 (PRMT5) enzyme of the methylosome complex as a MYPT1-binding protein uncovering the nuclear MYPT1-interactome of hepatocellular carcinoma cells. It is shown that PRMT5 is regulated by phosphorylation at Thr80 by RhoA-associated protein kinase and MP. Silencing of MYPT1 increased the level of the PRMT5-specific symmetric dimethylation on arginine residues of histone 2 A/4, a repressing gene expression mark, and it resulted in a global change in the expression of genes affecting cellular processes like growth, proliferation and cell death, also affecting the expression of the retinoblastoma protein and c-Myc. The phosphorylation of the MP inhibitory MYPT1T850 and the regulatory PRMT5T80 residues as well as the symmetric dimethylation of H2A/4 were elevated in human hepatocellular carcinoma and in other types of cancers. These changes correlated positively with the grade and state of the tumors. Our results suggest the tumor suppressor role of MP via inhibition of PRMT5 thereby regulating gene expression through histone arginine dimethylation. PMID:28074910

  20. Novel protein kinase C inhibitors: alpha-terthiophene derivatives.

    PubMed

    Kim, D S; Ashendel, C L; Zhou, Q; Chang, C T; Lee, E S; Chang, C J

    1998-10-06

    A series of alpha-terthiophene derivatives were prepared and their protein kinase C inhibitory activity were evaluated. The aldehyde derivatives were most potent inhibitors (IC50 < 1 microM). alpha-Terthiophene monoaldehyde was inactive in the inhibitions of protein kinase A, mitogen activated protein kinase and protein tyrosine kinase.

  1. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2004-10-12

    The present invention relates to 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  2. Schizosaccharomyces pombe AGC family kinase Gad8p forms a conserved signaling module with TOR and PDK1-like kinases

    PubMed Central

    Matsuo, Tomohiko; Kubo, Yoshiya; Watanabe, Yoshinori; Yamamoto, Masayuki

    2003-01-01

    The TOR protein is a phosphoinositide kinase-related kinase widely conserved among eukaryotes. Fission yeast tor1 encodes an ortholog of TOR, which is required for sexual development and growth under stressed conditions. We isolated gad8, which encodes a Ser/Thr kinase of the AGC family, as a high-copy suppressor of the sterility of a tor1 mutant. Disruption of gad8 caused phenotypes similar to those of tor1 disruption. Gad8p was less phosphorylated and its kinase activity was undetectable in tor1Δ cells. Three amino acid residues corresponding to conserved phosphorylation sites in the AGC family kinases, namely Thr387 in the activation loop, Ser527 in the turn motif and Ser546 in the hydrophobic motif, were important for the kinase activity of Gad8p. Tor1p was responsible for the phosphorylation of Ser527 and Ser546, whereas Ksg1p, a PDK1-like kinase, appeared to phosphorylate Thr387 directly. Altogether, Tor1p, Ksg1p and Gad8p appear to constitute a signaling module for sexual development and growth under stressed conditions in fission yeast, which resembles the mTOR–PDK1–S6K1 system in mammals and may represent a basic signaling module ubiquitous in eukaryotes. PMID:12805221

  3. Purification and characterization of a thylakoid protein kinase

    SciTech Connect

    Coughlan, S.J.; Hind, G.

    1986-01-01

    Control of state transitions in the thylakoid by reversible phosphorylation of the light-harvesting chlorophyll a/b protein complex of photosystem II (LHC-II) is modulated by a kinase. The kinase catalyzing this phosphorylation is associated with the thylakoid membrane, and is regulated by the redox state of the plastoquinone pool. The isolation and partial purification from spinach thylakoids of two protein kinases (CPK1, CPK2) of apparent molecular masses 25 kDa and 38 kDa has been reported. Neither enzyme utilizes isolated LHC-II as a substrate. The partial purification of a third protein kinase (LHCK) which can utilize both lysine-rich histones (IIIs and Vs) and isolated LHC-II as substrate has now been purified to homogeneity and characterized by SDS-polyacrylamide gel electrophoresis as a 64 kDa peptide. From a comparison of the two isolation procedures we have concluded that CPK1 is indeed a protein kinase, but has a lower specific activity than that of LHCK. 8 refs., 4 figs.

  4. Dynamics driven allostery in protein kinases

    PubMed Central

    Kornev, Alexandr P.; Taylor, Susan S.

    2015-01-01

    Protein kinases have very dynamic structures and their functionality strongly depends on their dynamic state. Active kinases reveal a dynamic pattern with residues clustering into semirigid communities that move in µs-ms timescale. Previously detected hydrophobic spines serve as connectors between communities. Communities do not follow the traditional subdomain structure of the kinase core or its secondary structure elements. Instead they are organized around main functional units. Integration of the communities depends on the assembly of the hydrophobic spine and phosphorylation of the activation loop. Single mutations can significantly disrupt the dynamic infrastructure and thereby interfere with long distance allosteric signaling that propagates throughout the whole molecule. Dynamics is proposed to be the underlying mechanism for allosteric regulation in protein kinases. PMID:26481499

  5. Molecular systems pharmacology: isoelectric focusing signature of protein kinase Cδ provides an integrated measure of its modulation in response to ligands.

    PubMed

    Kedei, Noemi; Chen, Jin-Qiu; Herrmann, Michelle A; Telek, Andrea; Goldsmith, Paul K; Petersen, Mark E; Keck, Gary E; Blumberg, Peter M

    2014-06-26

    Protein kinase C (PKC), a validated therapeutic target for cancer chemotherapy, provides a paradigm for assessing structure-activity relations, where ligand binding has multiple consequences for a target. For PKC, ligand binding controls not only PKC activation and multiple phosphorylations but also subcellular localization, affecting subsequent signaling. Using a capillary isoelectric focusing immunoassay system, we could visualize a high resolution isoelectric focusing signature of PKCδ upon stimulation by ligands of the phorbol ester and bryostatin classes. Derivatives that possessed different physicochemical characteristics and induced different patterns of biological response generated different signatures. Consistent with different patterns of PKCδ localization as one factor linked to these different signatures, we found different signatures for activated PKCδ from the nuclear and non-nuclear fractions. We conclude that the capillary isoelectric focusing immunoassay system may provide a window into the integrated consequences of ligand binding and thus afford a powerful platform for compound development.

  6. Oncoprotein protein kinase antibody kit

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2008-12-23

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

  7. Paradoxical stimulation and inhibition by protein kinase C modulating agents of lipopolysaccharide evoked production of tumour necrosis factor in human monocytes.

    PubMed Central

    Coffey, R G; Weakland, L L; Alberts, V A

    1992-01-01

    Human blood monocytes were activated by bacterial lipopolysaccharide endotoxin (LPS) (10 ng/ml) for cytotoxicity of WEHI-164 mouse fibrosarcoma cells, determined by release of 51Cr from WEHI-164 tumour cells incubated with monocyte supernatants. The chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) augmented LPS-induced cytotoxicity but had no effect alone. FMLP but not LPS stimulated phospholipase C (PLC), determined by the release of [3H]inositol phosphates. Addition of tumour promoter and protein kinase C stimulant, phorbol-12-myristate-13-acetate (PMA) at concentrations of 3 x 10(-10) M to 3 x 10(-9) M, resulted in an augmentation of 30-200% in LPS-evoked cytotoxicity. The effects of FMLP and PMA, like the effect of LPS alone, were completely blocked by antibody to recombinant human tumour necrosis factor-alpha (TNF-alpha), indicating that cytotoxicity induced by LPS, FMLP, and PMA were due solely to TNF release. Concentrations of PMA greater than 3 x 10(-9) M caused inhibition of TNF release. Okadaic acid (20 ng/ml), an inhibitor of phosphatases I and IIa, augmented the effects of LPS and the stimulatory effects of low levels of PMA, suggesting that phosphorylation was important in the actions of both LPS and PMA. The effects of LPS and of low levels of PMA were augmented by the protein kinase C (PKC) inhibitors H-7 (10-30 microM), staurosporine (2-10 nM) and calphostin C (0.1 microM). Higher concentrations of the inhibitors prevented LPS-evoked TNF release and its augmentation by low levels of PMA. However, they did not prevent the inhibition by high levels of PMA. One possible explanation for the results is that different isozymes of PKC may mediate the stimulatory as compared to the inhibitory effects of PKC on TNF production. PMID:1628900

  8. Protein Kinase A: A Master Kinase of Granulosa Cell Differentiation

    PubMed Central

    Puri, Pawan; Little-Ihrig, Lynda; Chandran, Uma; Law, Nathan C.; Hunzicker-Dunn, Mary; Zeleznik, Anthony J.

    2016-01-01

    Activation of protein kinase A (PKA) by follicle stimulating hormone (FSH) transduces the signal that drives differentiation of ovarian granulosa cells (GCs). An unresolved question is whether PKA is sufficient to initiate the complex program of GC responses to FSH. We compared signaling pathways and gene expression profiles of GCs stimulated with FSH or expressing PKA-CQR, a constitutively active mutant of PKA. Both FSH and PKA-CQR stimulated the phosphorylation of proteins known to be involved in GC differentiation including CREB, ß-catenin, AKT, p42/44 MAPK, GAB2, GSK-3ß, FOXO1, and YAP. In contrast, FSH stimulated the phosphorylation of p38 MAP kinase but PKA-CQR did not. Microarray analysis revealed that 85% of transcripts that were up-regulated by FSH were increased to a comparable extent by PKA-CQR and of the transcripts that were down-regulated by FSH, 76% were also down-regulated by PKA-CQR. Transcripts regulated similarly by FSH and PKA-CQR are involved in steroidogenesis and differentiation, while transcripts more robustly up-regulated by PKA-CQR are involved in ovulation. Thus, PKA, under the conditions of our experimental approach appears to function as a master upstream kinase that is sufficient to initiate the complex pattern of intracellular signaling pathway and gene expression profiles that accompany GC differentiation. PMID:27324437

  9. Protein kinase Calpha activation by RET: evidence for a negative feedback mechanism controlling RET tyrosine kinase.

    PubMed

    Andreozzi, Francesco; Melillo, Rosa Marina; Carlomagno, Francesca; Oriente, Francesco; Miele, Claudia; Fiory, Francesca; Santopietro, Stefania; Castellone, Maria Domenica; Beguinot, Francesco; Santoro, Massimo; Formisano, Pietro

    2003-05-15

    We have studied the role of protein kinase C (PKC) in signaling of the RET tyrosine kinase receptor. By using a chimeric receptor (E/R) in which RET kinase can be tightly controlled by the addition of epidermal growth factor (EGF), we have found that RET triggering induces a strong increase of PKCalpha, PKCdelta and PKCzeta activity and that PKCalpha, not PKCdelta and PKCzeta, forms a ligand-dependent protein complex with E/R. We have identified tyrosine 1062 in the RET carboxyl-terminal tail as the docking site for PKCalpha. Block of PKC activity by bisindolylmaleimide or chronic phorbol esters treatment decreased EGF-induced serine/threonine phosphorylation of E/R, while it caused a similarly sized increase of EGF-induced E/R tyrosine kinase activity and mitogenic signaling. Conversely, acute phorbol esters treatment, which promotes PKC activity, increased the levels of E/R serine/threonine phosphorylation and significantly decreased its phosphotyrosine content. A threefold reduction of tyrosine phosphorylation levels of the constitutively active RET/MEN2A oncoprotein was observed upon coexpression with PKCalpha. We conclude that RET binds to and activates PKCalpha. PKCalpha, in turn, causes RET phosphorylation and downregulates RET tyrosine kinase and downstream signaling, thus functioning as a negative feedback loop to modulate RET activity.

  10. AMP-activated protein kinase--an archetypal protein kinase cascade?

    PubMed

    Hardie, D G; MacKintosh, R W

    1992-10-01

    Mammalian AMP-activated protein kinase is the central component of a protein kinase cascade which inactivates three key enzymes involved in the synthesis or release of free fatty acids and cholesterol inside the cell. The kinase cascade is activated by elevation of AMP, and perhaps also by fatty acid and cholesterol metabolites. The system may fulfil a protective function, preventing damage caused by depletion of ATP or excessive intracellular release of free lipids, a type of stress response. Recent evidence suggests that it may have been in existence for at least a billion years, since a very similar protein kinase cascade is present in higher plants. This system therefore represents an early eukaryotic protein kinase cascade, which is unique in that it is regulated by intracellular metabolites rather than extracellular signals or cell cycle events.

  11. Flavonoid baicalein modulates H2O2-induced mitogen-activated protein kinases activation and cell death in SK-N-MC cells.

    PubMed

    Moslehi, Maryam; Meshkini, Azadeh; Yazdanparast, Razieh

    2012-05-01

    It is believed that ROS-induced oxidative stress triggers numerous signaling pathways which are involved in neurodegenerative diseases, including Alzheimer's disease. To find the effective drugs for neurodegenerative diseases, the deep delve into molecular mechanisms underlie these diseases is necessary. In the current study, we investigated the effects of flavonoid baicalein on H(2)O(2)-induced oxidative stress and cell death in SK-N-MC cells. Our results revealed that the treatment of SK-N-MC cells with H(2)O(2) led to a decrease in cell viability through phosphorylation and activation of extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs) pathways followed by increase in Bax/Bcl2 ratio and initiation of caspase-dependent apoptotic pathways. In addition, our results showed that the exposure of SK-N-MC cells to H(2)O(2) ended up in reduction of glutathione (GSH) levels of SK-N-MC cells via JNK/ERK-mediated down-regulation of γ-glutamyl-cysteine synthetase (γ-GCS) expression. Our results demonstrated that flavonoid baicalein protected against H(2)O(2)-induced cell death by inhibition of JNK/ERK pathways activation and other key molecules in apoptotic pathways, including blockage of Bax and caspase-9 activation, induction of Bcl-2 expression and prevention of cell death. Baicalein supported intracellular defense mechanisms through maintaining GSH levels in SK-N-MC cells by the removal of inhibition effects of JNK/ERK pathways from γ-GCS expression. In addition, baicalein attenuated lipid and protein peroxidation and intracellular reactive oxygen species in SK-N-MC cells. In accordance with these observations, baicalein can be a promising candidate in antioxidant therapy and designing of natural-based drug for ROS-induced neurodegenerative disorders.

  12. Phosphorylation of FE65 Ser610 by serum- and glucocorticoid-induced kinase 1 modulates Alzheimer's disease amyloid precursor protein processing

    PubMed Central

    Chow, Wan Ning Vanessa; Ngo, Jacky Chi Ki; Li, Wen; Chen, Yu Wai; Tam, Ka Ming Vincent; Chan, Ho Yin Edwin; Miller, Christopher C.J.; Lau, Kwok-Fai

    2015-01-01

    Alzheimer's disease (AD) is a fatal neurodegenerative disease affecting 36 million people worldwide. Genetic and biochemical research indicate that the excessive generation of amyloid-β peptide (Aβ) from amyloid precursor protein (APP), is a major part of AD pathogenesis. FE65 is a brain-enriched adaptor protein that binds to APP. However, the role of FE65 in APP processing and the mechanisms that regulate binding of FE65 to APP are not fully understood. In the present study, we show that serum- and glucocorticoid-induced kinase 1 (SGK1) phosphorylates FE65 on Ser610 and that this phosphorylation attenuates FE65 binding to APP. We also show that FE65 promotes amyloidogenic processing of APP and that FE65 Ser610 phosphorylation inhibits this effect. Furthermore, we found that the effect of FE65 Ser610 phosphorylation on APP processing is linked to a role of FE65 in metabolic turnover of APP via the proteasome. Thus FE65 influences APP degradation via the proteasome and phosphorylation of FE65 Ser610 by SGK1 regulates binding of FE65 to APP, APP turnover and processing. PMID:26188042

  13. Protein interactome analysis of 12 mitogen-activated protein kinase kinase kinase in rice using a yeast two-hybrid system.

    PubMed

    Singh, Raksha; Lee, Jae-Eun; Dangol, Sarmina; Choi, Jihyun; Yoo, Ran Hee; Moon, Jae Sun; Shim, Jae-Kyung; Rakwal, Randeep; Agrawal, Ganesh Kumar; Jwa, Nam-Soo

    2014-01-01

    The mitogen-activated protein kinase (MAPK) cascade is composed at least of MAP3K (for MAPK kinase kinase), MAP2K, and MAPK family modules. These components together play a central role in mediating extracellular signals to the cell and vice versa by interacting with their partner proteins. However, the MAP3K-interacting proteins remain poorly investigated in plants. Here, we utilized a yeast two-hybrid system and bimolecular fluorescence complementation in the model crop rice (Oryza sativa) to map MAP3K-interacting proteins. We identified 12 novel nonredundant interacting protein pairs (IPPs) representing 11 nonredundant interactors using 12 rice MAP3Ks (available as full-length cDNA in the rice KOME (http://cdna01.dna.affrc.go.jp/cDNA/) at the time of experimental design and execution) as bait and a rice seedling cDNA library as prey. Of the 12 MAP3Ks, only six had interacting protein partners. The established MAP3K interactome consisted of two kinases, three proteases, two forkhead-associated domain-containing proteins, two expressed proteins, one E3 ligase, one regulatory protein, and one retrotransposon protein. Notably, no MAP3K showed physical interaction with either MAP2K or MAPK. Seven IPPs (58.3%) were confirmed in vivo by bimolecular fluorescence complementation. Subcellular localization of 14 interactors, together involved in nine IPPs (75%) further provide prerequisite for biological significance of the IPPs. Furthermore, GO of identified interactors predicted their involvement in diverse physiological responses, which were supported by a literature survey. These findings increase our knowledge of the MAP3K-interacting proteins, help in proposing a model of MAPK modules, provide a valuable resource for developing a complete map of the rice MAPK interactome, and allow discussion for translating the interactome knowledge to rice crop improvement against environmental factors.

  14. eIF4A RNA Helicase Associates with Cyclin-Dependent Protein Kinase A in Proliferating Cells and Is Modulated by Phosphorylation1[OPEN

    PubMed Central

    Bush, Maxwell S.; Pierrat, Olivier; Nibau, Candida; Mikitova, Veronika; Zheng, Tao; Corke, Fiona M. K.; Mayberry, Laura K.; Browning, Karen S.

    2016-01-01

    Eukaryotic initiation factor 4A (eIF4A) is a highly conserved RNA-stimulated ATPase and helicase involved in the initiation of messenger RNA translation. Previously, we found that eIF4A interacts with cyclin-dependent kinase A (CDKA), the plant ortholog of mammalian CDK1. Here, we show that this interaction occurs only in proliferating cells where the two proteins coassociate with 5′-cap-binding protein complexes, eIF4F or the plant-specific eIFiso4F. CDKA phosphorylates eIF4A on a conserved threonine residue (threonine-164) within the RNA-binding motif 1b TPGR. In vivo, a phospho-null (APGR) variant of the Arabidopsis (Arabidopsis thaliana) eIF4A1 protein retains the ability to functionally complement a mutant (eif4a1) plant line lacking eIF4A1, whereas a phosphomimetic (EPGR) variant fails to complement. The phospho-null variant (APGR) rescues the slow growth rate of roots and rosettes, together with the ovule-abortion and late-flowering phenotypes. In vitro, wild-type recombinant eIF4A1 and its phospho-null variant both support translation in cell-free wheat germ extracts dependent upon eIF4A, but the phosphomimetic variant does not support translation and also was deficient in ATP hydrolysis and helicase activity. These observations suggest a mechanism whereby CDK phosphorylation has the potential to down-regulate eIF4A activity and thereby affect translation. PMID:27388680

  15. Label-Free Protein-RNA Interactome Analysis Identifies Khsrp Signaling Downstream of the p38/Mk2 Kinase Complex as a Critical Modulator of Cell Cycle Progression

    PubMed Central

    Schmitt, Anna; Riabinska, Arina; Thelen, Lisa; Peifer, Martin; Leeser, Uschi; Nuernberg, Peter; Altmueller, Janine; Gaestel, Matthias; Dieterich, Christoph; Reinhardt, H. Christian

    2015-01-01

    Growing evidence suggests a key role for RNA binding proteins (RBPs) in genome stability programs. Additionally, recent developments in RNA sequencing technologies, as well as mass-spectrometry techniques, have greatly expanded our knowledge on protein-RNA interactions. We here use full transcriptome sequencing and label-free LC/MS/MS to identify global changes in protein-RNA interactions in response to etoposide-induced genotoxic stress. We show that RBPs have distinct binding patterns in response to genotoxic stress and that inactivation of the RBP regulator module, p38/MK2, can affect the entire spectrum of protein-RNA interactions that take place in response to stress. In addition to validating the role of known RBPs like Srsf1, Srsf2, Elavl1 in the genotoxic stress response, we add a new collection of RBPs to the DNA damage response. We identify Khsrp as a highly regulated RBP in response to genotoxic stress and further validate its role as a driver of the G1/S transition through the suppression of Cdkn1aP21 transcripts. Finally, we identify KHSRP as an indicator of overall survival, as well as disease free survival in glioblastoma multiforme. PMID:25993413

  16. Protein kinase A regulates the osteogenic activity of Osterix.

    PubMed

    He, Siyuan; Choi, You Hee; Choi, Joong-Kook; Yeo, Chang-Yeol; Chun, ChangJu; Lee, Kwang Youl

    2014-10-01

    Osterix belongs to the SP gene family and is a core transcription factor responsible for osteoblast differentiation and bone formation. Activation of protein kinase A (PKA), a serine/threonine kinase, is essential for controlling bone formation and BMP-induced osteoblast differentiation. However, the relationship between Osterix and PKA is still unclear. In this report, we investigated the precise role of the PKA pathway in regulating Osterix during osteoblast differentiation. We found that PKA increased the protein level of Osterix; PKA phosphorylated Osterix, increased protein stability, and enhanced the transcriptional activity of Osterix. These results suggest that Osterix is a novel target of PKA, and PKA modulates osteoblast differentiation partially through the regulation of Osterix.

  17. Globular adiponectin inhibits ethanol-induced reactive oxygen species production through modulation of NADPH oxidase in macrophages: involvement of liver kinase B1/AMP-activated protein kinase pathway.

    PubMed

    Kim, Mi Jin; Nagy, Laura E; Park, Pil-Hoon

    2014-09-01

    Adiponectin, an adipokine predominantly secreted from adipocytes, has been shown to play protective roles against chronic alcohol consumption. Although excessive reactive oxygen species (ROS) production in macrophages is considered one of the critical events for ethanol-induced damage in various target tissues, the effect of adiponectin on ethanol-induced ROS production is not clearly understood. In the present study, we investigated the effect of globular adiponectin (gAcrp) on ethanol-induced ROS production and the potential mechanisms underlying these effects of gAcrp in macrophages. Here we demonstrated that gAcrp prevented ethanol-induced ROS production in both RAW 264.7 macrophages and primary murine peritoneal macrophages. Globular adiponectin also inhibited ethanol-induced activation of NADPH oxidase. In addition, gAcrp suppressed ethanol-induced increase in the expression of NADPH oxidase subunits, including Nox2 and p22(phox), via modulation of nuclear factor-κB pathway. Furthermore, pretreatment with compound C, a selective inhibitor of AMPK, or knockdown of AMPK by small interfering RNA restored suppression of ethanol-induced ROS production and Nox2 expression by gAcrp. Finally, we found that gAcrp treatment induced phosphorylation of liver kinase B1 (LKB1), an upstream signaling molecule mediating AMPK activation. Knockdown of LKB1 restored gAcrp-suppressed Nox2 expression, suggesting that LKB1/AMPK pathway plays a critical role in the suppression of ethanol-induced ROS production and activation of NADPH oxidase by gAcrp. Taken together, these results demonstrate that globular adiponectin prevents ethanol-induced ROS production, at least in part, via modulation of NADPH oxidase in macrophages. Further, LKB1/AMPK axis plays an important role in the suppression of ethanol-induced NADPH oxidase activation by gAcrp in macrophages.

  18. Caveolin-3 Overexpression Attenuates Cardiac Hypertrophy via Inhibition of T-type Ca2+ Current Modulated by Protein Kinase Cα in Cardiomyocytes*

    PubMed Central

    Markandeya, Yogananda S.; Phelan, Laura J.; Woon, Marites T.; Keefe, Alexis M.; Reynolds, Courtney R.; August, Benjamin K.; Hacker, Timothy A.; Roth, David M.; Patel, Hemal H.; Balijepalli, Ravi C.

    2015-01-01

    Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca2+ cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca2+ signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKCα and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca2+ current (ICa, T) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of ICa, T and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of ICa, T mediated by PKCα, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKCα-mediated increase in ICa, T and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy. PMID:26170457

  19. Problem-Solving Test: "In Vitro" Protein Kinase A Reaction

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

    Phosphorylation of proteins by protein kinases is an important mechanism in the regulation of protein activity. Among hundreds of protein kinases present in human cells, PKA, the first kinase discovered, belongs to the most important and best characterized group of these enzymes. The author presents an experiment that analyzes the "in vitro"…

  20. The protein interaction landscape of the human CMGC kinase group.

    PubMed

    Varjosalo, Markku; Keskitalo, Salla; Van Drogen, Audrey; Nurkkala, Helka; Vichalkovski, Anton; Aebersold, Ruedi; Gstaiger, Matthias

    2013-04-25

    Cellular information processing via reversible protein phosphorylation requires tight control of the localization, activity, and substrate specificity of protein kinases, which to a large extent is accomplished by complex formation with other proteins. Despite their critical role in cellular regulation and pathogenesis, protein interaction information is available for only a subset of the 518 human protein kinases. Here we present a global proteomic analysis of complexes of the human CMGC kinase group. In addition to subgroup-specific functional enrichment and modularity, the identified 652 high-confidence kinase-protein interactions provide a specific biochemical context for many poorly studied CMGC kinases. Furthermore, the analysis revealed a kinase-kinase subnetwork and candidate substrates for CMGC kinases. Finally, the presented interaction proteome uncovered a large set of interactions with proteins genetically linked to a range of human diseases, including cancer, suggesting additional routes for analyzing the role of CMGC kinases in controlling human disease pathways.

  1. Prevention of neuronal apoptosis by phorbol ester-induced activation of protein kinase C: blockade of p38 mitogen-activated protein kinase.

    PubMed

    Behrens, M M; Strasser, U; Koh, J Y; Gwag, B J; Choi, D W

    1999-01-01

    Consistent with previous studies on cell lines and non-neuronal cells, specific inhibitors of protein kinase C induced mouse primary cultured neocortical neurons to undergo apoptosis. To examine the complementary hypothesis that activating protein kinase C would attenuate neuronal apoptosis, the cultures were exposed for 1 h to phorbol-12-myristate-13-acetate, which activated protein kinase C as evidenced by downstream enhancement of the mitogen-activated protein kinase pathway. Exposure to phorbol-12-myristate-13-acetate, or another active phorbol ester, phorbol-12,13-didecanoate, but not to the inactive ester, 4alpha-phorbol-12,13-didecanoate, markedly attenuated neuronal apoptosis induced by serum deprivation. Phorbol-12-myristate-13-acetate also attenuated neuronal apoptosis induced by exposure to beta-amyloid peptide 1-42, or oxygen-glucose deprivation in the presence of glutamate receptor antagonists. The neuroprotective effects of phorbol-12-myristate-13-acetate were blocked by brief (non-toxic) concurrent exposure to the specific protein kinase C inhibitors, but not by a specific mitogen-activated protein kinase 1 inhibitor. Phorbol-12-myristate-13-acetate blocked the induction of p38 mitogen-activated protein kinase activity and specific inhibition of this kinase by SB 203580 attenuated serum deprivation-induced apoptosis. c-Jun N-terminal kinase 1 activity was high at rest and not modified by phorbol-12-myristate-13-acetate treatment. These data strengthen the idea that protein kinase C is a key modulator of several forms of central neuronal apoptosis, in part acting through inhibition of p38 mitogen-activated protein kinase regulated pathways.

  2. Mycobacterium tuberculosis Serine/Threonine Protein Kinases

    PubMed Central

    PRISIC, SLADJANA; HUSSON, ROBERT N.

    2014-01-01

    The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs). A similar number of two-component systems are also present, indicating that these two signal transduction mechanisms are both important in the adaptation of this bacterial pathogen to its environment. The M. tuberculosis phosphoproteome includes hundreds of Ser- and Thr-phosphorylated proteins that participate in all aspects of M. tuberculosis biology, supporting a critical role for the STPKs in regulating M. tuberculosis physiology. Nine of the STPKs are receptor type kinases, with an extracytoplasmic sensor domain and an intracellular kinase domain, indicating that these kinases transduce external signals. Two other STPKs are cytoplasmic and have regulatory domains that sense changes within the cell. Structural analysis of some of the STPKs has led to advances in our understanding of the mechanisms by which these STPKs are activated and regulated. Functional analysis has provided insights into the effects of phosphorylation on the activity of several proteins, but for most phosphoproteins the role of phosphorylation in regulating function is unknown. Major future challenges include characterizing the functional effects of phosphorylation for this large number of phosphoproteins, identifying the cognate STPKs for these phosphoproteins, and determining the signals that the STPKs sense. Ultimately, combining these STPK-regulated processes into larger, integrated regulatory networks will provide deeper insight into M. tuberculosis adaptive mechanisms that contribute to tuberculosis pathogenesis. Finally, the STPKs offer attractive targets for inhibitor development that may lead to new therapies for drug-susceptible and drug-resistant tuberculosis. PMID:25429354

  3. A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C.

    PubMed

    Luangwedchakarn, Voravich; Day, Noorbibi K; Hitchcock, Remi; Brown, Pam G; Lerner, Danica L; Rucker, Rajivi P; Cianciolo, George J; Good, Robert A; Haraguchi, Soichi

    2003-05-01

    CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.

  4. Protein kinase C-associated kinase regulates NF-κB activation through inducing IKK activation.

    PubMed

    Kim, Sang-Woo; Schifano, Matthew; Oleksyn, David; Jordan, Craig T; Ryan, Daniel; Insel, Richard; Zhao, Jiyong; Chen, Luojing

    2014-10-01

    Activation of the transcription factor NF-κB induced by extracellular stimuli requires IKKα and IKKβ kinase activity. How IKKα and IKKβ are activated by various upstream signaling molecules is not fully understood. We previously showed that protein kinase C-associated kinase (PKK, also known as DIK/RIP4), which belongs to the receptor-interacting protein (RIP) kinase family, mediates the B cell activating factor of the TNF family (BAFF)-induced NF-κB activation in diffuse large B cell lymphoma (DLBCL) cell lines. Here we have investigated the mechanism underlying NF-κB activation regulated by PKK. Our results suggest that PKK can activate both the classical and the alternative NF-κB activation pathways. PKK associates with IKKα and IKKβ in mammalian cells and induces activation of both IKKα and IKKβ via phosphorylation of their serine residues 176/180 and 177/181, respectively. Unlike other members of the RIP family that activate NF-κB through a kinase-independent pathway, PKK appears to activate IKK and NF-κB mainly in a kinase-dependent manner. Suppression of PKK expression by RNA interference inhibits phosphorylation of IKKα and IKKβ as well as activation of NF-κB in human cancer cell lines. Thus, PKK regulates NF-κB activation by modulating activation of IKKα and IKKβ in mammalian cells. We propose that PKK may provide a critical link between IKK activation and various upstream signaling cascades, and may represent a potential target for inhibiting abnormal NF-κB activation in human cancers.

  5. Mitogen-activated protein kinase 6 controls root growth in Arabidopsis by modulating Ca2+ -based Na+ flux in root cell under salt stress.

    PubMed

    Han, Shuan; Wang, Chi-wen; Wang, Wen-le; Jiang, Jing

    2014-03-01

    Little is known about the role of mitogen-activated protein kinase 6 (MPK6) in Na(+) toxicity and inhibition of root growth in Arabidopsis under NaCl stress. In this study, we found that root elongation in seedlings of the loss-of-function mutants mpk6-2 and mpk6-3 was less sensitive to NaCl or Na-glutamate, but not to KCl or mannitol, as compared with that of wild-type (WT) seedlings. The less sensitive characteristic was eliminated by adding the Ca(2+) chelator EGTA or the Ca(2+) channel inhibitor LaCl3, but not the Ca(2+) ionophore A23187. This suggested that the tolerance of mpk6 to Na(+) toxicity was Ca(2+)-dependent. We measured plasma membrane (PM) Na(+)-conducted currents (NCCs) in root cells. Increased concentrations of NaCl increased the inward NCCs while decreased the outward NCCs in WT root cells, attended by a positive shift in membrane potential. In mpk6 root cells, NaCl significantly increased outward but not inward NCCs, accompanied by a negative shift in membrane potential. That is, mpk6 decreased NaCl-induced the Na(+) accumulation by modifying PM Na(+) flux in root cells. Observations of aequorin luminescence revealed a NaCl-induced increase of cytosolic Ca(2+) in mpk6 root cells, resulting from PM Ca(2+) influx. An increase of cytosolic Ca(2+) was required to alleviate the NaCl-increased Na(+) content and Na(+)/K(+) ratio in mpk6 roots. Together, these results show that mpk6 accumulated less Na(+) in response to NaCl because of the increased cytosolic Ca(2+) level in root cells; thus, its root elongation was less inhibited than that of WT by NaCl.

  6. Modulation of Kv3.1b potassium channel phosphorylation in auditory neurons by conventional and novel protein kinase C isozymes.

    PubMed

    Song, Ping; Kaczmarek, Leonard K

    2006-06-02

    In fast-spiking neurons such as those in the medial nucleus of the trapezoid body (MNTB) in the auditory brainstem, Kv3.1 potassium channels are required for high frequency firing. The Kv3.1b splice variant of this channel predominates in the mature nervous system and is a substrate for phosphorylation by protein kinase C (PKC) at Ser-503. In resting neurons, basal phosphorylation at this site decreases Kv3.1 current, reducing neuronal ability to follow high frequency stimulation. We used a phospho-specific antibody to determine which PKC isozymes control serine 503 phosphorylation in Kv3.1b-tranfected cells and in auditory neurons in brainstem slices. By using isozyme-specific inhibitors, we found that the novel PKC-delta isozyme, together with the novel PKC-epsilon and conventional PKCs, contributed to the basal phosphorylation of Kv3.1b in MNTB neurons. In contrast, only PKC-epsilon and conventional PKCs mediate increases in phosphorylation produced by pharmacological activation of PKC in MNTB neurons or by metabotropic glutamate receptor activation in Kv3.1/mGluR1-cotransfected cells. We also measured the time course of dephosphorylation and recovery of basal phosphorylation of Kv3.1b following brief high frequency electrical stimulation of the trapezoid body, and we determined that the recovery process is mediated by both novel PKC-delta and PKC-epsilon isozymes and by conventional PKCs. The association between Kv3.1b and PKC isozymes was confirmed by reciprocal coimmunoprecipitation of Kv3.1b with multiple PKC isozymes. Our results suggest that the Kv3.1b channel is regulated by both conventional and novel PKC isozymes and that novel PKC-delta contributes specifically to the maintenance of basal phosphorylation in auditory neurons.

  7. Mining protein kinases regulation using graphical models.

    PubMed

    Chen, Qingfeng; Chen, Yi-Ping Phoebe

    2011-03-01

    Abnormal kinase activity is a frequent cause of diseases, which makes kinases a promising pharmacological target. Thus, it is critical to identify the characteristics of protein kinases regulation by studying the activation and inhibition of kinase subunits in response to varied stimuli. Bayesian network (BN) is a formalism for probabilistic reasoning that has been widely used for learning dependency models. However, for high-dimensional discrete random vectors the set of plausible models becomes large and a full comparison of all the posterior probabilities related to the competing models becomes infeasible. A solution to this problem is based on the Markov Chain Monte Carlo (MCMC) method. This paper proposes a BN-based framework to discover the dependency correlations of kinase regulation. Our approach is to apply the MCMC method to generate a sequence of samples from a probability distribution, by which to approximate the distribution. The frequent connections (edges) are identified from the obtained sampling graphical models. Our results point to a number of novel candidate regulation patterns that are interesting in biology and include inferred associations that were unknown.

  8. Mitogen Activated Protein kinase signal transduction pathways in the prostate

    PubMed Central

    Maroni, Paul D; Koul, Sweaty; Meacham, Randall B; Koul, Hari K

    2004-01-01

    The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy. PMID:15219238

  9. Crystal Structure of the Protein Kinase Domain of Yeast AMP-Activated Protein Kinase Snf1

    SciTech Connect

    Rudolph,M.; Amodeo, G.; Bai, Y.; Tong, L.

    2005-01-01

    AMP-activated protein kinase (AMPK) is a master metabolic regulator, and is an important target for drug development against diabetes, obesity, and other diseases. AMPK is a hetero-trimeric enzyme, with a catalytic ({alpha}) subunit, and two regulatory ({beta} and {gamma}) subunits. Here we report the crystal structure at 2.2 Angstrom resolution of the protein kinase domain (KD) of the catalytic subunit of yeast AMPK (commonly known as SNF1). The Snf1-KD structure shares strong similarity to other protein kinases, with a small N-terminal lobe and a large C-terminal lobe. Two negative surface patches in the structure may be important for the recognition of the substrates of this kinase.

  10. Kinase Inhibitor Screening Identifies Cyclin-Dependent Kinases and Glycogen Synthase Kinase 3 as Potential Modulators of TDP-43 Cytosolic Accumulation during Cell Stress.

    PubMed

    Moujalled, Diane; James, Janine L; Parker, Sarah J; Lidgerwood, Grace E; Duncan, Clare; Meyerowitz, Jodi; Nonaka, Takashi; Hasegawa, Masato; Kanninen, Katja M; Grubman, Alexandra; Liddell, Jeffrey R; Crouch, Peter J; White, Anthony R

    2013-01-01

    Abnormal processing of TAR DNA binding protein 43 (TDP-43) has been identified as a major factor in neuronal degeneration during amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). It is unclear how changes to TDP-43, including nuclear to cytosolic translocation and subsequent accumulation, are controlled in these diseases. TDP-43 is a member of the heterogeneous ribonucleoprotein (hnRNP) RNA binding protein family and is known to associate with cytosolic RNA stress granule proteins in ALS and FTLD. hnRNP trafficking and accumulation is controlled by the action of specific kinases including members of the mitogen-activated protein kinase (MAPK) pathway. However, little is known about how kinase pathways control TDP-43 movement and accumulation. In this study, we used an in vitro model of TDP-43-positve stress granule formation to screen for the effect of kinase inhibitors on TDP-43 accumulation. We found that while a number of kinase inhibitors, particularly of the MAPK pathways modulated both TDP-43 and the global stress granule marker, human antigen R (HuR), multiple inhibitors were more specific to TDP-43 accumulation, including inhibitors of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3 (GSK3). Close correlation was observed between effects of these inhibitors on TDP-43, hnRNP K and TIAR, but often with different effects on HuR accumulation. This may indicate a potential interaction between TDP-43, hnRNP K and TIAR. CDK inhibitors were also found to reverse pre-formed TDP-43-positive stress granules and both CDK and GSK3 inhibitors abrogated the accumulation of C-terminal TDP-43 (219-414) in transfected cells. Further studies are required to confirm the specific kinases involved and whether their action is through phosphorylation of the TDP-43 binding partner hnRNP K. This knowledge provides a valuable insight into the mechanisms controlling abnormal cytoplasmic TDP-43 accumulation and may herald new opportunities

  11. Protein Kinase C Isozyme in Mammary Carcinogenesis.

    DTIC Science & Technology

    1996-10-01

    11 A B Clone 72 Clone 34 AKAP 86 Clone 35H CInn 72 lonn 4 P 95 lone 351 9. 5J 4.4- 688 431 Clone 45 Clone 35F Clone 64 Annexln 1 Clone 45 Clone 35F...purified antibodies directed against a variety of PKC substrates and an A-kinase anchoring protein, AKAP 95. (B) PolyA+ mRNAs isolated from confluent cell

  12. MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase.

    PubMed Central

    Nakielny, S; Cohen, P; Wu, J; Sturgill, T

    1992-01-01

    A 'MAP kinase activator' was purified several thousand-fold from insulin-stimulated rabbit skeletal muscle, which resembled the 'activator' from nerve growth factor-stimulated PC12 cells in that it could be inactivated by incubation with protein phosphatase 2A, but not by protein tyrosine phosphatases and its apparent molecular mass was 45-50 kDa. In the presence of MgATP, 'MAP kinase activator' converted the normal 'wild-type' 42 kDa MAP kinase from an inactive dephosphorylated form to the fully active diphosphorylated species. Phosphorylation occurred on the same threonine and tyrosine residues which are phosphorylated in vivo in response to growth factors or phorbol esters. A mutant MAP kinase produced by changing a lysine at the active centre to arginine was phosphorylated in an identical manner by the 'MAP kinase activator', but no activity was generated. The results demonstrate that 'MAP kinase activator' is a protein kinase (MAP kinase kinase) and not a protein that stimulates the autophosphorylation of MAP kinase. MAP kinase kinase is the first established example of a protein kinase that can phosphorylate an exogenous protein on threonine as well as tyrosine residues. Images PMID:1318193

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

    PubMed

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

    2016-06-24

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

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

    PubMed

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

    2007-05-31

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

  15. Kinase Pathway Database: An Integrated Protein-Kinase and NLP-Based Protein-Interaction Resource

    PubMed Central

    Koike, Asako; Kobayashi, Yoshiyuki; Takagi, Toshihisa

    2003-01-01

    Protein kinases play a crucial role in the regulation of cellular functions. Various kinds of information about these molecules are important for understanding signaling pathways and organism characteristics. We have developed the Kinase Pathway Database, an integrated database involving major completely sequenced eukaryotes. It contains the classification of protein kinases and their functional conservation, ortholog tables among species, protein–protein, protein–gene, and protein–compound interaction data, domain information, and structural information. It also provides an automatic pathway graphic image interface. The protein, gene, and compound interactions are automatically extracted from abstracts for all genes and proteins by natural-language processing (NLP).The method of automatic extraction uses phrase patterns and the GENA protein, gene, and compound name dictionary, which was developed by our group. With this database, pathways are easily compared among species using data with more than 47,000 protein interactions and protein kinase ortholog tables. The database is available for querying and browsing at http://kinasedb.ontology.ims.u-tokyo.ac.jp/. PMID:12799355

  16. Cross-talk from β-adrenergic receptors modulates α2A-adrenergic receptor endocytosis in sympathetic neurons via protein kinase A and spinophilin.

    PubMed

    Cottingham, Christopher; Lu, Roujian; Jiao, Kai; Wang, Qin

    2013-10-04

    Inter-regulation of adrenergic receptors (ARs) via cross-talk is a long appreciated but mechanistically unclear physiological phenomenon. Evidence from the AR literature and our own extensive studies on regulation of α2AARs by the scaffolding protein spinophilin have illuminated a potential novel mechanism for cross-talk from β to α2ARs. In the present study, we have characterized a mode of endogenous AR cross-talk in native adrenergic neurons whereby canonical βAR-mediated signaling modulates spinophilin-regulated α2AAR endocytosis through PKA. Our findings demonstrate that co-activation of β and α2AARs, either by application of endogenous agonist or by simultaneous stimulation with distinct selective agonists, results in acceleration of endogenous α2AAR endocytosis in native neurons. We show that receptor-independent PKA activation by forskolin is sufficient to accelerate α2AAR endocytosis and that α2AAR stimulation alone drives accelerated endocytosis in spinophilin-null neurons. Endocytic response acceleration by β/α2AAR co-activation is blocked by PKA inhibition and lost in spinophilin-null neurons, consistent with our previous finding that spinophilin is a substrate for phosphorylation by PKA that disrupts its interaction with α2AARs. Importantly, we show that α2AR agonist-mediated α2AAR/spinophilin interaction is blocked by βAR co-activation in a PKA-dependent fashion. We therefore propose a novel mechanism for cross-talk from β to α2ARs, whereby canonical βAR-mediated signaling coupled to PKA activation results in phosphorylation of spinophilin, disrupting its interaction with α2AARs and accelerating α2AAR endocytic responses. This mechanism of cross-talk has significant implications for endogenous adrenergic physiology and for therapeutic targeting of β and α2AARs.

  17. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

    Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. PMID:26579181

  18. Structure of the pseudokinase-kinase domains from protein kinase TYK2 reveals a mechanism for Janus kinase (JAK) autoinhibition.

    PubMed

    Lupardus, Patrick J; Ultsch, Mark; Wallweber, Heidi; Bir Kohli, Pawan; Johnson, Adam R; Eigenbrot, Charles

    2014-06-03

    Janus kinases (JAKs) are receptor-associated multidomain tyrosine kinases that act downstream of many cytokines and interferons. JAK kinase activity is regulated by the adjacent pseudokinase domain via an unknown mechanism. Here, we report the 2.8-Å structure of the two-domain pseudokinase-kinase module from the JAK family member TYK2 in its autoinhibited form. We find that the pseudokinase and kinase interact near the kinase active site and that most reported mutations in cancer-associated JAK alleles cluster in or near this interface. Mutation of residues near the TYK2 interface that are analogous to those in cancer-associated JAK alleles, including the V617F and "exon 12" JAK2 mutations, results in increased kinase activity in vitro. These data indicate that JAK pseudokinases are autoinhibitory domains that hold the kinase domain inactive until receptor dimerization stimulates transition to an active state.

  19. Protein kinase C theta (PKCθ) modulates the ClC-1 chloride channel activity and skeletal muscle phenotype: a biophysical and gene expression study in mouse models lacking the PKCθ.

    PubMed

    Camerino, Giulia Maria; Bouchè, Marina; De Bellis, Michela; Cannone, Maria; Liantonio, Antonella; Musaraj, Kejla; Romano, Rossella; Smeriglio, Piera; Madaro, Luca; Giustino, Arcangela; De Luca, Annamaria; Desaphy, Jean-François; Camerino, Diana Conte; Pierno, Sabata

    2014-12-01

    In skeletal muscle, the resting chloride conductance (gCl), due to the ClC-1 chloride channel, controls the sarcolemma electrical stability. Indeed, loss-of-function mutations in ClC-1 gene are responsible of myotonia congenita. The ClC-1 channel can be phosphorylated and inactivated by protein kinases C (PKC), but the relative contribution of each PKC isoforms is unknown. Here, we investigated on the role of PKCθ in the regulation of ClC-1 channel expression and activity in fast- and slow-twitch muscles of mouse models lacking PKCθ. Electrophysiological studies showed an increase of gCl in the PKCθ-null mice with respect to wild type. Muscle excitability was reduced accordingly. However, the expression of the ClC-1 channel, evaluated by qRT-PCR, was not modified in PKCθ-null muscles suggesting that PKCθ affects the ClC-1 activity. Pharmacological studies demonstrated that although PKCθ appreciably modulates gCl, other isoforms are still active and concur to this role. The modification of gCl in PKCθ-null muscles has caused adaptation of the expression of phenotype-specific genes, such as calcineurin and myocyte enhancer factor-2, supporting the role of PKCθ also in the settings of muscle phenotype. Importantly, the lack of PKCθ has prevented the aging-related reduction of gCl, suggesting that its modulation may represent a new strategy to contrast the aging process.

  20. Probing the enzyme kinetics, allosteric modulation and activation of α1- and α2-subunit-containing AMP-activated protein kinase (AMPK) heterotrimeric complexes by pharmacological and physiological activators

    PubMed Central

    Rajamohan, Francis; Reyes, Allan R.; Frisbie, Richard K.; Hoth, Lise R.; Sahasrabudhe, Parag; Magyar, Rachelle; Landro, James A.; Withka, Jane M.; Caspers, Nicole L.; Calabrese, Matthew F.; Ward, Jessica; Kurumbail, Ravi G.

    2015-01-01

    AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that serves as a pleotropic regulator of whole body energy homoeostasis. AMPK exists as a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (β and γ), each present as multiple isoforms. In the present study, we compared the enzyme kinetics and allosteric modulation of six recombinant AMPK isoforms, α1β1γ1, α1β2γ1, α1β2γ3, α2β1γ1, α2β2γ1 and α2β2γ3 using known activators, A769662 and AMP. The α1-containing complexes exhibited higher specific activities and lower Km values for a widely used peptide substrate (SAMS) compared with α2-complexes. Surface plasmon resonance (SPR)-based direct binding measurements revealed biphasic binding modes with two distinct equilibrium binding constants for AMP, ADP and ATP across all isoforms tested. The α2-complexes were ∼25-fold more sensitive than α1-complexes to dephosphorylation of a critical threonine on their activation loop (pThr172/174). However, α2-complexes were more readily activated by AMP than α1-complexes. Compared with β1-containing heterotrimers, β2-containing AMPK isoforms are less sensitive to activation by A769662, a synthetic activator. These data demonstrate that ligand induced activation of AMPK isoforms may vary significantly based on their AMPK subunit composition. Our studies provide insights for the design of isoform-selective AMPK activators for the treatment of metabolic diseases. PMID:26635351

  1. Endothelial Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 Is Critical for Lymphatic Vascular Development and Function

    PubMed Central

    Guo, Chang-An; Danai, Laura V.; Yawe, Joseph C.; Gujja, Sharvari; Edwards, Yvonne J. K.

    2016-01-01

    The molecular mechanisms underlying lymphatic vascular development and function are not well understood. Recent studies have suggested a role for endothelial cell (EC) mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) in developmental angiogenesis and atherosclerosis. Here, we show that constitutive loss of EC Map4k4 in mice causes postnatal lethality due to chylothorax, suggesting that Map4k4 is required for normal lymphatic vascular function. Mice constitutively lacking EC Map4k4 displayed dilated lymphatic capillaries, insufficient lymphatic valves, and impaired lymphatic flow; furthermore, primary ECs derived from these animals displayed enhanced proliferation compared with controls. Yeast 2-hybrid analyses identified the Ras GTPase-activating protein Rasa1, a known regulator of lymphatic development and lymphatic endothelial cell fate, as a direct interacting partner for Map4k4. Map4k4 silencing in ECs enhanced basal Ras and extracellular signal-regulated kinase (Erk) activities, and primary ECs lacking Map4k4 displayed enhanced lymphatic EC marker expression. Taken together, these results reveal that EC Map4k4 is critical for lymphatic vascular development by regulating EC quiescence and lymphatic EC fate. PMID:27044870

  2. Mixed - Lineage Protein kinases (MLKs) in inflammation, metabolism, and other disease states.

    PubMed

    Craige, Siobhan M; Reif, Michaella M; Kant, Shashi

    2016-09-01

    Mixed lineage kinases, or MLKs, are members of the MAP kinase kinase kinase (MAP3K) family, which were originally identified among the activators of the major stress-dependent mitogen activated protein kinases (MAPKs), JNK and p38. During stress, the activation of JNK and p38 kinases targets several essential downstream substrates that react in a specific manner to the unique stressor and thus determine the fate of the cell in response to a particular challenge. Recently, the MLK family was identified as a specific modulator of JNK and p38 signaling in metabolic syndrome. Moreover, the MLK family of kinases appears to be involved in a very wide spectrum of disorders. This review discusses the newly identified functions of MLKs in multiple diseases including metabolic disorders, inflammation, cancer, and neurological diseases.

  3. Implications of mitogen-activated protein kinase signaling in glioma.

    PubMed

    Pandey, Vimal; Bhaskara, Vasantha Kumar; Babu, Phanithi Prakash

    2016-02-01

    Gliomas are the most common primary central nervous system tumors. Gliomas originate from astrocytes, oligodendrocytes, and neural stem cells or their precursors. According to WHO classification, gliomas are classified into four different malignant grades ranging from grade I to grade IV based on histopathological features and related molecular aberrations. The induction and maintenance of these tumors can be attributed largely to aberrant signaling networks. In this regard, the mitogen-activated protein kinase (MAPK) network has been widely studied and is reported to be severely altered in glial tumors. Mutations in MAPK pathways most frequently affect RAS and B-RAF in the ERK, c-Jun N-terminal kinase (JNK), and p38 pathways leading to malignant transformation. Also, it is linked to both inherited and sequential accumulations of mutations that control receptor tyrosine kinase (RTK)-activated signal transduction pathways, cell cycle growth arrest pathways, and nonresponsive cell death pathways. Genetic alterations that modulate RTK signaling can also alter several downstream pathways, including RAS-mediated MAP kinases along with JNK pathways, which ultimately regulate cell proliferation and cell death. The present review focuses on recent literature regarding important deregulations in the RTK-activated MAPK pathway during gliomagenesis and progression.

  4. Glycogen Synthase Kinase 3β Interaction Protein Functions as an A-kinase Anchoring Protein*

    PubMed Central

    Hundsrucker, Christian; Skroblin, Philipp; Christian, Frank; Zenn, Hans-Michael; Popara, Viola; Joshi, Mangesh; Eichhorst, Jenny; Wiesner, Burkhard; Herberg, Friedrich W.; Reif, Bernd; Rosenthal, Walter; Klussmann, Enno

    2010-01-01

    A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3β interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3β (glycogen synthase kinase 3β). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3β by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3β and thereby provides a mechanism for the integration of PKA and GSK3β signaling pathways. PMID:20007971

  5. Musk Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop.

    PubMed

    Camurdanoglu, B Z; Hrovat, C; Dürnberger, G; Madalinski, M; Mechtler, K; Herbst, R

    2016-09-26

    The neuromuscular junction (NMJ) forms when a motor neuron contacts a muscle fibre. A reciprocal exchange of signals initiates a cascade of signalling events that result in pre- and postsynaptic differentiation. At the centre of these signalling events stands muscle specific kinase (MuSK). MuSK activation, kinase activity and subsequent downstream signalling are crucial for NMJ formation as well as maintenance. Therefore MuSK kinase activity is tightly regulated to ensure proper NMJ development. We have identified a novel serine phosphorylation site at position 751 in MuSK that is increasingly phosphorylated upon agrin stimulation. S751 is also phosphorylated in muscle tissue and its phosphorylation depends on MuSK kinase activity. A phosphomimetic mutant of S751 increases MuSK kinase activity in response to non-saturating agrin concentrations . In addition, basal MuSK and AChR phosphorylation as well as AChR cluster size are increased. We believe that the phosphorylation of S751 provides a novel mechanism to relief the autoinhibition of the MuSK activation loop. Such a lower autoinhibition could foster or stabilize MuSK kinase activation, especially during stages when no or low level of agrin are present. Phosphorylation of S751 might therefore represent a novel mechanism to modulate MuSK kinase activity during prepatterning or NMJ maintenance.

  6. Photoinduced structural changes to protein kinase A

    NASA Astrophysics Data System (ADS)

    Rozinek, Sarah C.; Thomas, Robert J.; Brancaleon, Lorenzo

    2014-03-01

    The importance of porphyrins in organisms is underscored by the ubiquitous biological and biochemical functions that are mediated by these compounds and by their potential biomedical and biotechnological applications. Protoporphyrin IX (PPIX) is the precursor to heme and has biomedical applications such as its use as a photosensitizer in phototherapy and photodetection of cancer. Among other applications, our group has demonstrated that low-irradiance exposure to laser irradiation of PPIX, Fe-PPIX, or meso-tetrakis (4-sulfonatophenyl) porphyrin (TSPP) non-covalently docked to a protein causes conformational changes in the polypeptide. Such approach can have remarkable consequences in the study of protein structure/function relationship and can be used to prompt non-native protein properties. Therefore we have investigated protein kinase A (PKA), a more relevant protein model towards the photo-treatment of cancer. PKA's enzymatic functions are regulated by the presence of cyclic adenosine monophosphate for intracellular signal transduction involved in, among other things, stimulation of transcription, tumorigenesis in Carney complex and migration of breast carcinoma cells. Since phosphorylation is a necessary step in some cancers and inflammatory diseases, inhibiting the protein kinase, and therefore phosphorylation, may serve to treat these diseases. Changes in absorption, steady-state fluorescence, and fluorescence lifetime indicate: 1) both TSPP and PPIX non-covalently bind to PKA where they maintain photoreactivity; 2) absorptive photoproduct formation occurs only when PKA is bound to TSPP and irradiated; and 3) PKA undergoes secondary structural changes after irradiation with either porphyrin bound. These photoinduced changes could affect the protein's enzymatic and signaling capabilities.

  7. The CDC2-related kinase PITALRE is the catalytic subunit of active multimeric protein complexes.

    PubMed Central

    Garriga, J; Mayol, X; Graña, X

    1996-01-01

    PITALRE is a human protein kinase identified by means of its partial sequence identity to the cell division cycle regulatory kinase CDC2. Immunopurified PITALRE protein complexes exhibit an in vitro kinase activity that phosphorylates the retinoblastoma protein, suggesting that PITALRE catalyses this phosphorylation reaction. However, the presence of other kinases in the immunopurified complex could not be ruled out. In the present work, an inactive mutant of the PITALRE kinase has been used to demonstrate that PITALRE is the catalytic subunit responsible for the PITALRE-complex-associated kinase activity, Ectopic overexpression of PITALRE did not increase the total PITALRE kinase activity in the cell, suggesting that PITALRE is regulated by limiting cellular factor(s). Characterization of the PITALRE-containing protein complexes indicated that most of the cellular PITALRE protein exists as a subunit in at least two different active multimeric complexes. Although monomeric PITALRE is also active in vitro, PITALRE present in multimeric complexes exhibits several-fold higher activity than monomeric PITALRE. In addition, overexpression of PITALRE demonstrated the existence of two new associated proteins of approx. 48 and 98 kDa. Altogether these results suggest that, in contrast to the situation with cyclin-dependent kinases, monomeric PITALRE is active, and that association with other proteins modulates its activity and/or its ability to recognize substrates in vivo. PMID:8870681

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

    PubMed

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

    2009-04-01

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

  9. Protein Kinases in Mammary Gland Development and Carcinogenesis

    DTIC Science & Technology

    1999-09-01

    differ among CaM kinase family members include their subcellular localization , regulation by autophosphorylation, and regulation by other proteins. In...addition, CaM kinases have unique amino- and carboxyl- terminal domains that contribute to kinase-specific differences in subcellular localization ...chromosomal localization of Punc, a calcium/calmodulin-dependent protein kinase, (Submitted). 14. Hennings, H., Glick, A., Lowry, D., Krsmanovic, L

  10. Cytoskeletal Modulation of Lipid Interactions Regulates Lck Kinase Activity*

    PubMed Central

    Chichili, Gurunadh R.; Cail, Robert C.; Rodgers, William

    2012-01-01

    The actin cytoskeleton promotes clustering of proteins associated with cholesterol-dependent rafts, but its effect on lipid interactions that form and maintain rafts is not understood. We addressed this question by determining the effect of disrupting the cytoskeleton on co-clustering of dihexadecyl-(C16)-anchored DiO and DiI, which co-enrich in ordered lipid environments such as rafts. Co-clustering was assayed by fluorescence resonance energy transfer (FRET) in labeled T cells, where rafts function in the phosphoregulation of the Src family kinase Lck. Our results show that probe co-clustering was sensitive to depolymerization of actin filaments with latrunculin B (Lat B), inhibition of myosin II with blebbistatin, and treatment with neomycin to sequester phosphatidylinositol 4,5-bisphosphate. Cytoskeletal effects on lipid interactions were not restricted to order-preferring label because co-clustering of C16-anchored DiO with didodecyl (C12)-anchored DiI, which favors disordered lipids, was also reduced by Lat B and blebbistatin. Furthermore, conditions that disrupted probe co-clustering resulted in activation of Lck. These data show that the cytoskeleton globally modulates lipid interactions in the plasma membrane, and this property maintains rafts that function in Lck regulation. PMID:22613726

  11. Protein kinase C alpha-dependent phosphorylation of Golgi proteins.

    PubMed

    Radau, B; Otto, A; Müller, E C; Westermann, P

    2000-07-01

    Golgi-enriched membranes were phosphorylated in order to understand the mechanism for protein kinase C (PKC) regulation of exocytic vesicle formation at the trans-Golgi network. Two of the main PKC substrates were identified as MARCKS and Mac-MARCKS by two-dimensional electrophoresis (2-DE) and mass spectrometric sequencing. Annexin IV and profilin I, two other Golgi-associated proteins--although known as in vitro PKC substrates--were not phosphorylated in the Golgi-bound state.

  12. Modulation of basal nitric oxide-dependent cyclic-GMP production by ambient glucose, myo-inositol, and protein kinase C in SH-SY5Y human neuroblastoma cells.

    PubMed Central

    Shindo, H; Thomas, T P; Larkin, D D; Karihaloo, A K; Inada, H; Onaya, T; Stevens, M J; Greene, D A

    1996-01-01

    Defective tissue perfusion and nitric oxide production and altered myo-inositol metabolism and protein kinase C activation have been invoked in the pathogenesis of diabetic complications including neuropathy. The precise cellular compartmentalization and mechanistic interrelationships of these abnormalities remain obscure, and nitric oxide possesses both neurotransmitter and vasodilator activity. Therefore the effects of ambient glucose and myo-inositol on nitric oxide-dependent cGMP production and protein kinase C activity were studied in SH-SY5Y human neuroblastoma cells, a cell culture model for peripheral cholinergic neurons. D-Glucose lowered cellular myo-inositol content, phosphatidylinositol synthesis, and phosphorylation of an endogenous protein kinase C substrate, and specifically reduced nitric oxide-dependent cGMP production a time- and dose-dependent manner with an apparent IC50 of approximately 30 mM. The near maximal decrease in cGMP induced by 50 mM D-glucose was corrected by the addition of protein kinase C agonists or 500 microM myo-inositol to the culture medium, and was reproduced by protein kinase C inhibition or downregulation, or by myo-inositol deficient medium. Sodium nitroprusside increased cGMP in a dose-dependent fashion, with low concentrations (1 microM) counteracting the effects of 50 mM D-glucose or protein kinase C inhibition. The demonstration that elevated D-glucose diminishes basal nitric oxide-dependent cGMP production by myo-inositol depletion and protein kinase C inhibition in peripheral cholinergic neurons provides a potential metabolic basis for impaired nitric oxide production, nerve blood flow, and nerve impulse conduction in diabetes. PMID:8609230

  13. Protein Kinases in Zucchini (Characterization of Calcium-Requiring Plasma Membrane Kinases).

    PubMed Central

    Verhey, S. D.; Gaiser, J. C.; Lomax, T. L.

    1993-01-01

    Using an in situ phosphorylation assay with zucchini (Cucurbita pepo L. cv Dark Green) seedling tissue, we have identified numerous polypeptides that are capable of acting as protein kinases. Total protein preparations from different organs contain different kinase profiles, but all are within the range of 55 to 70 kD. At least four kinases are associated with highly purified plasma membranes from etiolated zucchini hypocotyls. The major phosphorylated polypeptides from plasma membranes range in apparent molecular mass from 58 to 68 kD. The plasma membrane kinases are activated by micromolar concentrations of calcium and phosphorylate serine, and, to a lesser extent, threonine residues. These characteristics are similar to those of a soluble calcium-dependent protein kinase that has been purified to homogeneity from soybean suspension cultures. Three of the zucchini plasma membrane kinases share antigenic epitopes with the soluble soybean kinase. The presence of kinase activity at different apparent molecular masses may be indicative of separate kinases with similar characteristics. The zucchini hypocotyl protein kinases are not removed from plasma membrane vesicles by 0.5 M NaCl/5 mM ethylenediaminetetraacetate or by detergent concentrations below the critical micelle concentration of two types of detergent. This indicates that the plasma membrane protein kinases are tightly associated with the membrane in zucchini seedlings. PMID:12231949

  14. Protein kinase Cepsilon is important for migration of neuroblastoma cells

    PubMed Central

    Stensman, Helena; Larsson, Christer

    2008-01-01

    Background Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility. Methods PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot. Results Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Gö6976 inhibited migration while an inhibitor of PKCβ isoforms did not have an effect. Downregulation of PKCε, but not of PKCα or PKCδ, with siRNA led to a suppression of both basal and TPA-stimulated migration. Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKCε. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS. Conclusion PKCε is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKCε but they may be involved in TPA-mediated migration. PMID:19077250

  15. Increased dietary protein attenuates C-reactive protein and creatine kinase responses to exercise-induced energy deficit

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We determined if dietary protein (P) modulates responses of C-reactive protein (CRP) and creatine kinase (CK), biomarkers of inflammation and muscle damage, during exercise-induced energy deficit (DEF). Thirteen healthy men (22 +/- 1 y, VO2peak 60 +/- 2 ml.kg-1.min-1) balanced energy expenditure (EE...

  16. Effects of selective inhibition of protein kinase C, cyclic AMP-dependent protein kinase, and Ca(2+)-calmodulin-dependent protein kinase on neurite development in cultured rat hippocampal neurons.

    PubMed

    Cabell, L; Audesirk, G

    1993-06-01

    parameter that was measured (including viability). These results suggest that these three protein kinases selectively modulate different aspects of neurite development. The university of effects caused by calmodulin inhibition make it impossible to determine if there are specific targets of calmodulin action involved in neurite development. Finally, our data indicate that some superficially similar characteristics of neuronal differentiation, such as neurite initiation and branching, may be controlled by quite different molecular mechanisms.

  17. Phosphoregulators: Protein Kinases and Protein Phosphatases of Mouse

    PubMed Central

    Forrest, Alistair R.R.; Ravasi, Timothy; Taylor, Darrin; Huber, Thomas; Hume, David A.; Grimmond, Sean

    2003-01-01

    With the completion of the human and mouse genome sequences, the task now turns to identifying their encoded transcripts and assigning gene function. In this study, we have undertaken a computational approach to identify and classify all of the protein kinases and phosphatases present in the mouse gene complement. A nonredundant set of these sequences was produced by mining Ensembl gene predictions and publicly available cDNA sequences with a panel of InterPro domains. This approach identified 561 candidate protein kinases and 162 candidate protein phosphatases. This cohort was then analyzed using TribeMCL protein sequence similarity clustering followed by CLUSTALV alignment and hierarchical tree generation. This approach allowed us to (1) distinguish between true members of the protein kinase and phosphatase families and enzymes of related biochemistry, (2) determine the structure of the families, and (3) suggest functions for previously uncharacterized members. The classifications obtained by this approach were in good agreement with previous schemes and allowed us to demonstrate domain associations with a number of clusters. Finally, we comment on the complementary nature of cDNA and genome-based gene detection and the impact of the FANTOM2 transcriptome project. PMID:12819143

  18. Targeting of calcium/calmodulin-dependent protein kinase II.

    PubMed Central

    Colbran, Roger J

    2004-01-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) has diverse roles in virtually all cell types and it is regulated by a plethora of mechanisms. Local changes in Ca2+ concentration drive calmodulin binding and CaMKII activation. Activity is controlled further by autophosphorylation at multiple sites, which can generate an autonomously active form of the kinase (Thr286) or can block Ca2+/calmodulin binding (Thr305/306). The regulated actions of protein phosphatases at these sites also modulate downstream signalling from CaMKII. In addition, CaMKII targeting to specific subcellular microdomains appears to be necessary to account for the known signalling specificity, and targeting is regulated by Ca2+/calmodulin and autophosphorylation. The present review focuses on recent studies revealing the diversity of CaMKII interactions with proteins localized to neuronal dendrites. Interactions with various subunits of the NMDA (N-methyl-D-aspartate) subtype of glutamate receptor have attracted the most attention, but binding of CaMKII to cytoskeletal and several other regulatory proteins has also been reported. Recent reports describing the molecular basis of each interaction and their potential role in the normal regulation of synaptic transmission and in pathological situations are discussed. These studies have revealed fundamental regulatory mechanisms that are probably important for controlling CaMKII functions in many cell types. PMID:14653781

  19. Protein kinase C activity in boar sperm.

    PubMed

    Teijeiro, J M; Marini, P E; Bragado, M J; Garcia-Marin, L J

    2017-03-01

    Male germ cells undergo different processes within the female reproductive tract to successfully fertilize the oocyte. These processes are triggered by different extracellular stimuli leading to activation of protein phosphorylation. Protein kinase C (PKC) is a key regulatory enzyme in signal transduction mechanisms involved in many cellular processes. Studies in boar sperm demonstrated a role for PKC in the intracellular signaling involved in motility and cellular volume regulation. Experiments using phorbol 12-myristate 13-acetate (PMA) showed increases in the Serine/Threonine phosphorylation of substrates downstream of PKC in boar sperm. In order to gain knowledge about those cellular processes regulated by PKC, we evaluate the effects of PMA on boar sperm motility, lipid organization of plasma membrane, integrity of acrosome membrane and sperm agglutination. Also, we investigate the crosstalk between PKA and PKC intracellular pathways in spermatozoa from this species. The results presented here reveal a participation of PKC in sperm motility regulation and membrane fluidity changes, which is probably associated to acrosome reaction and to agglutination. Also, we show the existence of a hierarchy in the kinases pathway. Previous works on boar sperm suggest a pathway in which PKA is positioned upstream to PKC and this new results support such model.

  20. Genomic analysis of the eukaryotic protein kinase superfamily: a perspective

    PubMed Central

    Hanks, Steven K

    2003-01-01

    Protein kinases with a conserved catalytic domain make up one of the largest 'superfamilies' of eukaryotic proteins and play many key roles in biology and disease. Efforts to identify and classify all the members of the eukaryotic protein kinase superfamily have recently culminated in the mining of essentially complete human genome data. PMID:12734000

  1. The Roles of Protein Kinases in Learning and Memory

    ERIC Educational Resources Information Center

    Giese, Karl Peter; Mizuno, Keiko

    2013-01-01

    In the adult mammalian brain, more than 250 protein kinases are expressed, but only a few of these kinases are currently known to enable learning and memory. Based on this information it appears that learning and memory-related kinases either impact on synaptic transmission by altering ion channel properties or ion channel density, or regulate…

  2. Interferon-gamma and transforming growth factor-beta modulate the activation of mitogen-activated protein kinases and tumor necrosis factor-alpha production induced by Fc gamma-receptor stimulation in murine macrophages.

    PubMed

    Rose, D M; Winston, B W; Chan, E D; Riches, D W; Henson, P M

    1997-09-08

    Engagement of receptors for the Fc region of IgG (Fc gamma R) can activate a variety of biological responses in macrophages, and these responses can be modulated either positively or negatively by co-stimulation with a variety of agents including cytokines such as interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta). We have previously demonstrated that Fc gamma R crosslinking activates the mitogen-activated protein kinase (MAPK) family members p42MAPK, p38, and JNK. Herein, we examined the modulatory effect of IFN-gamma, TGF-beta, and platelet-activating factor (PAF) on Fc gamma R-induced MAPK activation in murine macrophages. Fc gamma R-induced activation of p42MAPK and JNK was augmented nearly two-fold by pretreatment with IFN-gamma. Conversely, TGF-beta pretreatment suppressed Fc gamma R-induced activation of p42MAPK, JNK, and p38. These modulatory effects of IFN-gamma and TGF-beta on MAPK activation correlated with changes in Fc gamma R-stimulated TNF-alpha production by these two cytokines.

  3. The fruit of Acanthopanax senticosus (Rupr. et Maxim.) Harms improves insulin resistance and hepatic lipid accumulation by modulation of liver adenosine monophosphate-activated protein kinase activity and lipogenic gene expression in high-fat diet-fed obese mice.

    PubMed

    Saito, Tetsuo; Nishida, Miyako; Saito, Masafumi; Tanabe, Akari; Eitsuka, Takahiro; Yuan, Shi-Hua; Ikekawa, Nobuo; Nishida, Hiroshi

    2016-10-01

    Obesity-associated insulin resistance is a major risk factor for most metabolic diseases, including dyslipidemia and type 2 diabetes. Acanthopanax senticosus (Rupr. et Maxim.) Harms (Goka) root has been used in traditional Chinese medicine for treatment of diabetes and other conditions; however, little is known about the effects of Goka fruit (GF). Goka fruit is rich in anthocyanin, which has beneficial effects on obesity and insulin resistance via activation of adenosine monophosphate-activated protein kinase (AMPK). We hypothesized that GF can improve obesity-associated insulin resistance. The aim of the present study was to investigate whether GF improves insulin resistance in high-fat diet (HFD)-induced obese mice. High-fat diet mice treated with GF (500 and 1000 mg/kg) for 12 weeks showed an improved glucose tolerance and insulin sensitivity, as well as reduced plasma insulin and liver lipid accumulation. Moreover, GF administration to HFD mice resulted in down-regulation of fatty acid synthase expression and up-regulation of cholesterol 7-alpha-hydroxylase expression in the liver. Notably, AMPK phosphorylation in the liver increased after GF administration. In summary, GF supplementation improved obesity-associated insulin resistance and hepatic lipid accumulation through modulation of AMPK activity and lipid metabolism-associated gene expression.

  4. Phosphate Concentration and the Putative Sensor Kinase Protein CckA Modulate Cell Lysis and Release of the Rhodobacter capsulatus Gene Transfer Agent

    PubMed Central

    Westbye, A. B.; Leung, M. M.; Florizone, S. M.; Taylor, T. A.; Johnson, J. A.; Fogg, P. C.

    2013-01-01

    The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a bacteriophage-like genetic element with the sole known function of horizontal gene transfer. Homologues of RcGTA genes are present in many members of the alphaproteobacteria and may serve an important role in microbial evolution. Transcription of RcGTA genes is induced as cultures enter the stationary phase; however, little is known about cis-active sequences. In this work, we identify the promoter of the first gene in the RcGTA structural gene cluster. Additionally, gene transduction frequency depends on the growth medium, and the reason for this is not known. We report that millimolar concentrations of phosphate posttranslationally inhibit the lysis-dependent release of RcGTA from cells in both a complex medium and a defined medium. Furthermore, we found that cell lysis requires the genes rcc00555 and rcc00556, which were expressed and studied in Escherichia coli to determine their predicted functions as an endolysin and holin, respectively. Production of RcGTA is regulated by host systems, including a putative histidine kinase, CckA, and we found that CckA is required for maximal expression of rcc00555 and for maturation of RcGTA to yield gene transduction-functional particles. PMID:23995641

  5. Artemisia asiatica Nakai Attenuates the Expression of Proinflammatory Mediators in Stimulated Macrophages Through Modulation of Nuclear Factor-κB and Mitogen-Activated Protein Kinase Pathways.

    PubMed

    Kim, Eun-Kyung; Tang, Yujiao; Cha, Kwang-Suk; Choi, Heeri; Lee, Chun Bok; Yoon, Jin-Hwan; Kim, Sang Bae; Kim, Jong-Shik; Kim, Jong Moon; Han, Weon Cheol; Choi, Suck-Jun; Lee, Sangmin; Choi, Eun-Ju; Kim, Sang-Hyun

    2015-08-01

    The present study aimed to examine the anti-inflammatory effects and potential mechanism of action of Artemisia asiatica Nakai (A. asiatica Nakai) extract in activated murine macrophages. A. asiatica Nakai extract showed dose-dependent suppression of lipopolysaccharide (LPS)-induced nitric oxide, inducible nitric oxide synthase, and cyclooxygenase-2 activity. It also showed dose-dependent inhibition of nuclear factor-κB (NF-κB) translocation from the cytosol to the nucleus and as an inhibitor of NF-κB-alpha phosphorylation. The extract's inhibitory effects were found to be mediated through NF-κB inhibition and phosphorylation of extracellular signal-regulated kinase 1/2 and p38 in LPS-stimulated J774A.1 murine macrophages, suggesting a potential mechanism for the anti-inflammatory activity of A. asiatica Nakai. To our knowledge, this is the first report of the anti-inflammatory effects of A. asiatica Nakai on J774A.1 murine macrophages; these results may help develop functional foods possessing an anti-inflammatory activity.

  6. Artemisia asiatica Nakai Attenuates the Expression of Proinflammatory Mediators in Stimulated Macrophages Through Modulation of Nuclear Factor-κB and Mitogen-Activated Protein Kinase Pathways

    PubMed Central

    Kim, Eun-Kyung; Tang, Yujiao; Cha, Kwang-Suk; Choi, Heeri; Lee, Chun Bok; Yoon, Jin-Hwan; Kim, Sang Bae; Kim, Jong-Shik; Kim, Jong Moon; Han, Weon Cheol; Choi, Suck-Jun; Lee, Sangmin; Choi, Eun-Ju; Kim, Sang-Hyun

    2015-01-01

    Abstract The present study aimed to examine the anti-inflammatory effects and potential mechanism of action of Artemisia asiatica Nakai (A. asiatica Nakai) extract in activated murine macrophages. A. asiatica Nakai extract showed dose-dependent suppression of lipopolysaccharide (LPS)-induced nitric oxide, inducible nitric oxide synthase, and cyclooxygenase-2 activity. It also showed dose-dependent inhibition of nuclear factor-κB (NF-κB) translocation from the cytosol to the nucleus and as an inhibitor of NF-κB-alpha phosphorylation. The extract's inhibitory effects were found to be mediated through NF-κB inhibition and phosphorylation of extracellular signal-regulated kinase 1/2 and p38 in LPS-stimulated J774A.1 murine macrophages, suggesting a potential mechanism for the anti-inflammatory activity of A. asiatica Nakai. To our knowledge, this is the first report of the anti-inflammatory effects of A. asiatica Nakai on J774A.1 murine macrophages; these results may help develop functional foods possessing an anti-inflammatory activity. PMID:26061361

  7. The nuts and bolts of AGC protein kinases.

    PubMed

    Pearce, Laura R; Komander, David; Alessi, Dario R

    2010-01-01

    The AGC kinase subfamily of protein kinases contains 60 members, including PKA, PKG and PKC. The family comprises some intensely examined protein kinases (such as Akt, S6K, RSK, MSK, PDK1 and GRK) as well as many less well-studied enzymes (such as SGK, NDR, LATS, CRIK, SGK494, PRKX, PRKY and MAST). Research has shed new light onto the architecture and regulatory mechanisms of these kinases. In addition, AGC kinases mediate diverse and important cellular functions, and their mutation and/or dysregulation contributes to the pathogenesis of many human diseases, including cancer and diabetes.

  8. Protein kinase A contributes to the negative control of Snf1 protein kinase in Saccharomyces cerevisiae.

    PubMed

    Barrett, LaKisha; Orlova, Marianna; Maziarz, Marcin; Kuchin, Sergei

    2012-02-01

    Snf1 protein kinase regulates responses to glucose limitation and other stresses. Snf1 activation requires phosphorylation of its T-loop threonine by partially redundant upstream kinases (Sak1, Tos3, and Elm1). Under favorable conditions, Snf1 is turned off by Reg1-Glc7 protein phosphatase. The reg1 mutation causes increased Snf1 activation and slow growth. To identify new components of the Snf1 pathway, we searched for mutations that, like snf1, suppress reg1 for the slow-growth phenotype. In addition to mutations in genes encoding known pathway components (SNF1, SNF4, and SAK1), we recovered "fast" mutations, designated fst1 and fst2. Unusual morphology of the mutants in the Σ1278b strains employed here helped us identify fst1 and fst2 as mutations in the RasGAP genes IRA1 and IRA2. Cells lacking Ira1, Ira2, or Bcy1, the negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA), exhibited reduced Snf1 pathway activation. Conversely, Snf1 activation was elevated in cells lacking the Gpr1 sugar receptor, which contributes to PKA signaling. We show that the Snf1-activating kinase Sak1 is phosphorylated in vivo on a conserved serine (Ser1074) within an ideal PKA motif. However, this phosphorylation alone appears to play only a modest role in regulation, and Sak1 is not the only relevant target of the PKA pathway. Collectively, our results suggest that PKA, which integrates multiple regulatory inputs, could contribute to Snf1 regulation under various conditions via a complex mechanism. Our results also support the view that, like its mammalian counterpart, AMP-activated protein kinase (AMPK), yeast Snf1 participates in metabolic checkpoint control that coordinates growth with nutrient availability.

  9. KinasePhos: a web tool for identifying protein kinase-specific phosphorylation sites.

    PubMed

    Huang, Hsien-Da; Lee, Tzong-Yi; Tzeng, Shih-Wei; Horng, Jorng-Tzong

    2005-07-01

    KinasePhos is a novel web server for computationally identifying catalytic kinase-specific phosphorylation sites. The known phosphorylation sites from public domain data sources are categorized by their annotated protein kinases. Based on the profile hidden Markov model, computational models are learned from the kinase-specific groups of the phosphorylation sites. After evaluating the learned models, the model with highest accuracy was selected from each kinase-specific group, for use in a web-based prediction tool for identifying protein phosphorylation sites. Therefore, this work developed a kinase-specific phosphorylation site prediction tool with both high sensitivity and specificity. The prediction tool is freely available at http://KinasePhos.mbc.nctu.edu.tw/.

  10. Phosphoproteomic Analysis of Protein Kinase C Signaling in Saccharomyces cerevisiae Reveals Slt2 Mitogen-activated Protein Kinase (MAPK)-dependent Phosphorylation of Eisosome Core Components*

    PubMed Central

    Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J.; Molina, María

    2013-01-01

    The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1–cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module. PMID:23221999

  11. CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases but we made the surprising observation that soybean CDPK' and several Arabidopsis isoforms (AtCPK4 and AtCPK34) could also autophosphorylate on tyrosine residues. In studies with His6-GmCDPK', we ide...

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

    PubMed

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inês C R; Willige, Björn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

    2014-06-19

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

  13. Solubilized placental membrane protein inhibits insulin receptor tyrosine kinase activity

    SciTech Connect

    Strout, H.V. Jr.; Slater, E.E.

    1987-05-01

    Regulation of insulin receptor (IR) tyrosine kinase (TK) activity may be important in modulating insulin action. Utilizing an assay which measures IR phosphorylation of angiotensin II (AII), the authors investigated whether fractions of TX-100 solubilized human placental membranes inhibited IR dependent AII phosphorylation. Autophosphorylated IR was incubated with membrane fractions before the addition of AII, and kinase inhibition measured by the loss of TSP incorporated in AII. An inhibitory activity was detected which was dose, time, and temperature dependent. The inhibitor was purified 200-fold by sequential chromatography on wheat germ agglutinin, DEAE, and hydroxyapatite. This inhibitory activity was found to correlate with an 80 KD protein which was electroeluted from preparative slab gels and rabbit antiserum raised. Incubation of membrane fractions with antiserum before the IRTK assay immunoprecipitated the inhibitor. Protein immunoblots of crude or purified fractions revealed only the 80 KD protein. Since IR autophosphorylation is crucial to IRTK activity, the authors investigated the state of IR autophosphorylation after treatment with inhibitor; no change was detected by phosphoamino acid analysis.

  14. Conformation-selective ATP-competitive inhibitors control regulatory interactions and noncatalytic functions of mitogen-activated protein kinases.

    PubMed

    Hari, Sanjay B; Merritt, Ethan A; Maly, Dustin J

    2014-05-22

    Most potent protein kinase inhibitors act by competing with ATP to block the phosphotransferase activity of their targets. However, emerging evidence demonstrates that ATP-competitive inhibitors can affect kinase interactions and functions in ways beyond blocking catalytic activity. Here, we show that stabilizing alternative ATP-binding site conformations of the mitogen-activated protein kinases (MAPKs) p38α and Erk2 with ATP-competitive inhibitors differentially, and in some cases divergently, modulates the abilities of these kinases to interact with upstream activators and deactivating phosphatases. Conformation-selective ligands are also able to modulate Erk2's ability to allosterically activate the MAPK phosphatase DUSP6, highlighting how ATP-competitive ligands can control noncatalytic kinase functions. Overall, these studies underscore the relationship between the ATP-binding and regulatory sites of MAPKs and provide insight into how ATP-competitive ligands can be designed to confer graded control over protein kinase function.

  15. Identification of intracellular receptor proteins for activated protein kinase C.

    PubMed Central

    Mochly-Rosen, D; Khaner, H; Lopez, J

    1991-01-01

    Protein kinase C (PKC) translocates from the cytosol to the particulate fraction on activation. This activation-induced translocation of PKC is thought to reflect PKC binding to the membrane lipids. However, immunological and biochemical data suggest that PKC may bind to proteins in the cytoskeletal elements in the particulate fraction and in the nuclei. Here we describe evidence for the presence of intracellular receptor proteins that bind activated PKC. Several proteins from the detergent-insoluble material of the particulate fraction bound PKC in the presence of phosphatidylserine and calcium; binding was further increased with the addition of diacylglycerol. Binding of PKC to two of these proteins was concentration-dependent, saturable, and specific, suggesting that these binding proteins are receptors for activated C-kinase, termed here "RACKs." PKC binds to RACKs via a site on PKC distinct from the substrate binding site. We suggest that binding to RACKs may play a role in activation-induced translocation of PKC. Images PMID:1850844

  16. AKAP-Lbc nucleates a protein kinase D activation scaffold.

    PubMed

    Carnegie, Graeme K; Smith, F Donelson; McConnachie, George; Langeberg, Lorene K; Scott, John D

    2004-09-24

    The transmission of cellular signals often proceeds through multiprotein complexes where enzymes are positioned in proximity to their upstream activators and downstream substrates. In this report we demonstrate that the A-kinase anchoring protein AKAP-Lbc assembles an activation complex for the lipid-dependent enzyme protein kinase D (PKD). Using a combination of biochemical, enzymatic, and immunofluorescence techniques, we show that the anchoring protein contributes to PKD activation in two ways: it recruits an upstream kinase PKCeta and coordinates PKA phosphorylation events that release activated protein kinase D. Thus, AKAP-Lbc synchronizes PKA and PKC activities in a manner that leads to the activation of a third kinase. This configuration illustrates the utility of kinase anchoring as a mechanism to constrain the action of broad-spectrum enzymes.

  17. Structural investigation of protein kinase C inhibitors.

    PubMed

    Barak, D; Shibata, M; Rein, R

    1991-01-01

    The phospholipid and Ca2+ dependent protein kinase (PKC) plays an essential role in a variety of cellular events. Inhibition of PKC was shown to arrest growth in tumor cell cultures making it a target for possible antitumor therapy. Calphostins are potent inhibitors of PKC with high affinity for the enzyme regulatory site. Structural characteristics of calphostins, which confer the inhibitory activity, are investigated by comparing their optimized structures with the existing models for PKC activation. The resulting model of inhibitory activity assumes interaction with two out of the three electrostatic interaction sites postulated for activators. The model shows two sites of hydrophobic interaction and enables the inhibitory activity of gossypol to be accounted for.

  18. Structural investigation of protein kinase C inhibitors

    NASA Technical Reports Server (NTRS)

    Barak, D.; Shibata, M.; Rein, R.

    1991-01-01

    The phospholipid and Ca2+ dependent protein kinase (PKC) plays an essential role in a variety of cellular events. Inhibition of PKC was shown to arrest growth in tumor cell cultures making it a target for possible antitumor therapy. Calphostins are potent inhibitors of PKC with high affinity for the enzyme regulatory site. Structural characteristics of calphostins, which confer the inhibitory activity, are investigated by comparing their optimized structures with the existing models for PKC activation. The resulting model of inhibitory activity assumes interaction with two out of the three electrostatic interaction sites postulated for activators. The model shows two sites of hydrophobic interaction and enables the inhibitory activity of gossypol to be accounted for.

  19. Protein-protein interactions of tandem affinity purification-tagged protein kinases in rice.

    PubMed

    Rohila, Jai S; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald; Dardick, Chris; Canlas, Patrick; Xu, Xia; Gribskov, Michael; Kanrar, Siddhartha; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E

    2006-04-01

    Forty-one rice cDNAs encoding protein kinases were fused to the tandem affinity purification (TAP) tag and expressed in transgenic rice plants. The TAP-tagged kinases and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by mass spectrometry. Ninety-five percent of the TAP-tagged kinases were recovered. Fifty-six percent of the TAP-tagged kinases were found to interact with other rice proteins. A number of these interactions were consistent with known protein complexes found in other species, validating the TAP-tag method in rice plants. Phosphorylation sites were identified on four of the kinases that interacted with either 14-3-3 proteins or cyclins.

  20. Multiple Kinases Involved in the Nicotinic Modulation of Gamma Oscillations in the Rat Hippocampal CA3 Area

    PubMed Central

    Wang, JianGang; He, XiaoLong; Guo, Fangli; Cheng, XiangLin; Wang, Yali; Wang, XiaoFang; Feng, ZhiWei; Vreugdenhil, Martin; Lu, ChengBiao

    2017-01-01

    Neuronal synchronization at gamma band frequency (20–80 Hz, γ oscillations) is closely associated with higher brain function, such as learning, memory and attention. Nicotinic acetylcholine receptors (nAChRs) are highly expressed in the hippocampus, and modulate hippocampal γ oscillations, but the intracellular mechanism underlying such modulation remains elusive. We explored multiple kinases by which nicotine can modulate γ oscillations induced by kainate in rat hippocampal area CA3 in vitro. We found that inhibitors of cyclic AMP dependent kinase (protein kinase A, PKA), protein kinase C (PKC), N-methyl-D-aspartate receptor (NMDA) receptors, Phosphoinositide 3-kinase (PI3K) and extracellular signal-related kinases (ERK), each individually could prevent the γ oscillation-enhancing effect of 1 μM nicotine, whereas none of them affected baseline γ oscillation strength. Inhibition of the serine/threonine kinase Akt increased baseline γ oscillations and partially blocked its nicotinic enhancement. We propose that the PKA-NMDAR-PI3K-ERK pathway modifies cellular properties required for the nicotinic enhancement of γ oscillations, dependent on a PKC-ERK mediated pathway. These signaling pathways provide clues for restoring γ oscillations in pathological conditions affecting cognition. The suppression of γ oscillations at 100 μM nicotine was only dependent on PKA-NMDAR activation and may be due to very high intracellular calcium levels. PMID:28321180

  1. Prostaglandin E2 negatively regulates AMP-activated protein kinase via protein kinase A signaling pathway.

    PubMed

    Funahashi, Koji; Cao, Xia; Yamauchi, Masako; Kozaki, Yasuko; Ishiguro, Naoki; Kambe, Fukushi

    2009-01-01

    We investigated possible involvement of prostaglandin (PG) E2 in regulation of AMP-activated protein kinase (AMPK). When osteoblastic MG63 cells were cultured in serum-deprived media, Thr-172 phosphorylation of AMPK alpha-subunit was markedly increased. Treatment of the cells with PGE2 significantly reduced the phosphorylation. Ser-79 phosphorylation of acetyl-CoA carboxylase, a direct target for AMPK, was also reduced by PGE2. On the other hand, PGE2 reciprocally increased Ser-485 phosphorylation of the alpha-subunit that could be associated with inhibition of AMPK activity. These effects of PGE2 were mimicked by PGE2 receptor EP2 and EP4 agonists and forskolin, but not by EP1 and EP3 agonists, and the effects were suppressed by an adenylate cyclase inhibitor SQ22536 and a protein kinase A inhibitor H89. Additionally, the PGE2 effects were duplicated in primary calvarial osteoblasts. Together, the present study demonstrates that PGE2 negatively regulates AMPK activity via activation of protein kinase A signaling pathway.

  2. Protein Kinase CK2: Intricate Relationships within Regulatory Cellular Networks.

    PubMed

    Nuñez de Villavicencio-Diaz, Teresa; Rabalski, Adam J; Litchfield, David W

    2017-03-05

    Protein kinase CK2 is a small family of protein kinases that has been implicated in an expanding array of biological processes. While it is widely accepted that CK2 is a regulatory participant in a multitude of fundamental cellular processes, CK2 is often considered to be a constitutively active enzyme which raises questions about how it can be a regulatory participant in intricately controlled cellular processes. To resolve this apparent paradox, we have performed a systematic analysis of the published literature using text mining as well as mining of proteomic databases together with computational assembly of networks that involve CK2. These analyses reinforce the notion that CK2 is involved in a broad variety of biological processes and also reveal an extensive interplay between CK2 phosphorylation and other post-translational modifications. The interplay between CK2 and other post-translational modifications suggests that CK2 does have intricate roles in orchestrating cellular events. In this respect, phosphorylation of specific substrates by CK2 could be regulated by other post-translational modifications and CK2 could also have roles in modulating other post-translational modifications. Collectively, these observations suggest that the actions of CK2 are precisely coordinated with other constituents of regulatory cellular networks.

  3. Protein Kinase CK2: Intricate Relationships within Regulatory Cellular Networks

    PubMed Central

    Nuñez de Villavicencio-Diaz, Teresa; Rabalski, Adam J.; Litchfield, David W.

    2017-01-01

    Protein kinase CK2 is a small family of protein kinases that has been implicated in an expanding array of biological processes. While it is widely accepted that CK2 is a regulatory participant in a multitude of fundamental cellular processes, CK2 is often considered to be a constitutively active enzyme which raises questions about how it can be a regulatory participant in intricately controlled cellular processes. To resolve this apparent paradox, we have performed a systematic analysis of the published literature using text mining as well as mining of proteomic databases together with computational assembly of networks that involve CK2. These analyses reinforce the notion that CK2 is involved in a broad variety of biological processes and also reveal an extensive interplay between CK2 phosphorylation and other post-translational modifications. The interplay between CK2 and other post-translational modifications suggests that CK2 does have intricate roles in orchestrating cellular events. In this respect, phosphorylation of specific substrates by CK2 could be regulated by other post-translational modifications and CK2 could also have roles in modulating other post-translational modifications. Collectively, these observations suggest that the actions of CK2 are precisely coordinated with other constituents of regulatory cellular networks. PMID:28273877

  4. Dihydrotestosterone differentially modulates the mitogen-activated protein kinase and the phosphoinositide 3-kinase/Akt pathways through the nuclear and novel membrane androgen receptor in C6 cells.

    PubMed

    Gatson, Joshua W; Kaur, Paramjit; Singh, Meharvan

    2006-04-01

    Androgens such as dihydrotestosterone (DHT) are known to exert their effects through the activation of intracellular receptors that regulate the transcription of target genes. Alternatively, nongenomic mechanisms, including the activation of such signaling pathways as the MAPK pathways, have been described. It is unclear, however, whether this latter mechanism of action is mediated by the classical androgen receptor (AR) or some alternative mechanism. In this study, using a glial cell model (C6 cells) that we found to express the AR, we identified that DHT increased the phosphorylation of both ERK and Akt, key effectors of the neuroprotection-associated MAPK and phosphoinositide 3-kinase signaling pathways, respectively, and ERK phosphorylation was blocked by the AR antagonist, flutamide. In contrast, the membrane-impermeable, BSA-conjugated androgen (DHT-BSA) caused a dose-dependent suppression of ERK and Akt phosphorylation, suggesting the existence of a novel membrane-associated AR that mediates this opposite effect on neuroprotective signaling. This is also supported by the observation of DHT-displaceable binding sites on the cell surface of live C6 cells. Collectively, these data support the existence of a novel membrane-associated AR in glial cells and argue for the existence of two, potentially competing, pathways in a given cell or tissue. This mutual antagonism was supported by the ability of DHT-BSA to attenuate DHT-induced ERK phosphorylation. Thus, depending on the predominance of one receptor mechanism over another, the outcome of androgen treatment may be very different and, as such, could help explain existing discrepancies as to whether androgens are protective or damage inducing.

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  6. Association of protein kinase Cmu with type II phosphatidylinositol 4-kinase and type I phosphatidylinositol-4-phosphate 5-kinase.

    PubMed

    Nishikawa, K; Toker, A; Wong, K; Marignani, P A; Johannes, F J; Cantley, L C

    1998-09-04

    Protein kinase Cmu (PKCmu), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952-960), and its mechanism of regulation is not yet clear. Here we show that PKCmu forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCmu between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCmu failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCmu-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCmu including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCmu acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.

  7. Polyphyllin G induce apoptosis and autophagy in human nasopharyngeal cancer cells by modulation of AKT and mitogen-activated protein kinase pathways in vitro and in vivo.

    PubMed

    Chen, Jui-Chieh; Hsieh, Ming-Ju; Chen, Chih-Jung; Lin, Jen-Tsun; Lo, Yu-Sheng; Chuang, Yi-Ching; Chien, Su-Yu; Chen, Mu-Kuan

    2016-10-25

    Polyphyllin G (also call polyphyllin VII), extract from rhizomes of Paris yunnanensis Franch, has been demonstrated to have strong anticancer activities in a wide variety of human cancer cell lines. Previous studies found that Polyphyllin G induced apoptotic cell death in human hepatoblastoma cancer and lung cancer cells. However, the underlying mechanisms of autophagy in human nasopharyngeal carcinoma (NPC) remain unclear. In this study, Polyphyllin G can potently induced apoptosis dependent on the activations of caspase-8, -3, and -9 and the changes of Bcl-2, Bcl-xL and Bax protein expression in different human NPC cell lines (HONE-1 and NPC-039). The amount of both LC3-II and Beclin-1 was intriguingly increased suggest that autophagy was induced in Polyphyllin G-treated NPC cells. To further clarify whether Polyphyllin G-induced apoptosis and autophagy depended on AKT/ERK/JNK/p38 MAPK signaling pathways, cells were combined treated with AKT inhibitor (LY294002), ERK1/2 inhibitor (U0126), p38 MAPK inhibitor (SB203580), or JNK inhibitor (SP600125). These results demonstrated that Polyphyllin G induced apoptosis in NPC cells through activation of ERK, while AKT, p38 MAPK and JNK were responsible for Polyphyllin G-induced autophagy. Finally, an administration of Polyphyllin G effectively suppressed the tumor growth in the NPC carcinoma xenograft model in vivo. In conclusion, our results reveal that Polyphyllin G inhibits cell viability and induces apoptosis and autophagy in NPC cancer cells, suggesting that Polyphyllin G is an attractive candidate for tumor therapies. Polyphyllin G may promise candidate for development of antitumor drugs targeting nasopharyngeal carcinoma.

  8. Polyphyllin G induce apoptosis and autophagy in human nasopharyngeal cancer cells by modulation of AKT and mitogen-activated protein kinase pathways in vitro and in vivo

    PubMed Central

    Chen, Chih-Jung; Lin, Jen-Tsun; Lo, Yu-Sheng; Chuang, Yi-Ching; Chien, Su-Yu; Chen, Mu-Kuan

    2016-01-01

    Polyphyllin G (also call polyphyllin VII), extract from rhizomes of Paris yunnanensis Franch, has been demonstrated to have strong anticancer activities in a wide variety of human cancer cell lines. Previous studies found that Polyphyllin G induced apoptotic cell death in human hepatoblastoma cancer and lung cancer cells. However, the underlying mechanisms of autophagy in human nasopharyngeal carcinoma (NPC) remain unclear. In this study, Polyphyllin G can potently induced apoptosis dependent on the activations of caspase-8, -3, and -9 and the changes of Bcl-2, Bcl-xL and Bax protein expression in different human NPC cell lines (HONE-1 and NPC-039). The amount of both LC3-II and Beclin-1 was intriguingly increased suggest that autophagy was induced in Polyphyllin G-treated NPC cells. To further clarify whether Polyphyllin G-induced apoptosis and autophagy depended on AKT/ERK/JNK/p38 MAPK signaling pathways, cells were combined treated with AKT inhibitor (LY294002), ERK1/2 inhibitor (U0126), p38 MAPK inhibitor (SB203580), or JNK inhibitor (SP600125). These results demonstrated that Polyphyllin G induced apoptosis in NPC cells through activation of ERK, while AKT, p38 MAPK and JNK were responsible for Polyphyllin G-induced autophagy. Finally, an administration of Polyphyllin G effectively suppressed the tumor growth in the NPC carcinoma xenograft model in vivo. In conclusion, our results reveal that Polyphyllin G inhibits cell viability and induces apoptosis and autophagy in NPC cancer cells, suggesting that Polyphyllin G is an attractive candidate for tumor therapies. Polyphyllin G may promise candidate for development of antitumor drugs targeting nasopharyngeal carcinoma. PMID:27602962

  9. JAK tyrosine kinases promote hierarchical activation of Rho and Rap modules of integrin activation.

    PubMed

    Montresor, Alessio; Bolomini-Vittori, Matteo; Toffali, Lara; Rossi, Barbara; Constantin, Gabriela; Laudanna, Carlo

    2013-12-23

    Lymphocyte recruitment is regulated by signaling modules based on the activity of Rho and Rap small guanosine triphosphatases that control integrin activation by chemokines. We show that Janus kinase (JAK) protein tyrosine kinases control chemokine-induced LFA-1- and VLA-4-mediated adhesion as well as human T lymphocyte homing to secondary lymphoid organs. JAK2 and JAK3 isoforms, but not JAK1, mediate CXCL12-induced LFA-1 triggering to a high affinity state. Signal transduction analysis showed that chemokine-induced activation of the Rho module of LFA-1 affinity triggering is dependent on JAK activity, with VAV1 mediating Rho activation by JAKs in a Gαi-independent manner. Furthermore, activation of Rap1A by chemokines is also dependent on JAK2 and JAK3 activity. Importantly, activation of Rap1A by JAKs is mediated by RhoA and PLD1, thus establishing Rap1A as a downstream effector of the Rho module. Thus, JAK tyrosine kinases control integrin activation and dependent lymphocyte trafficking by bridging chemokine receptors to the concurrent and hierarchical activation of the Rho and Rap modules of integrin activation.

  10. Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

    SciTech Connect

    Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy; Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A.; Rhee, Kirsten; Tisdale, Sarah; Danial, Nika; Benarafa, Charaf; Orduna, Anonio; Anderson, Paul

    2010-10-22

    Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

  11. Protein kinase C phosphorylates AMP-activated protein kinase α1 Ser487

    PubMed Central

    Heathcote, Helen R.; Mancini, Sarah J.; Strembitska, Anastasiya; Jamal, Kunzah; Reihill, James A.; Palmer, Timothy M.; Gould, Gwyn W.; Salt, Ian P.

    2016-01-01

    The key metabolic regulator, AMP-activated protein kinase (AMPK), is reported to be down-regulated in metabolic disorders, but the mechanisms are poorly characterised. Recent studies have identified phosphorylation of the AMPKα1/α2 catalytic subunit isoforms at Ser487/491, respectively, as an inhibitory regulation mechanism. Vascular endothelial growth factor (VEGF) stimulates AMPK and protein kinase B (Akt) in cultured human endothelial cells. As Akt has been demonstrated to be an AMPKα1 Ser487 kinase, the effect of VEGF on inhibitory AMPK phosphorylation in cultured primary human endothelial cells was examined. Stimulation of endothelial cells with VEGF rapidly increased AMPKα1 Ser487 phosphorylation in an Akt-independent manner, without altering AMPKα2 Ser491 phosphorylation. In contrast, VEGF-stimulated AMPKα1 Ser487 phosphorylation was sensitive to inhibitors of protein kinase C (PKC) and PKC activation using phorbol esters or overexpression of PKC-stimulated AMPKα1 Ser487 phosphorylation. Purified PKC and Akt both phosphorylated AMPKα1 Ser487 in vitro with similar efficiency. PKC activation was associated with reduced AMPK activity, as inhibition of PKC increased AMPK activity and phorbol esters inhibited AMPK, an effect lost in cells expressing mutant AMPKα1 Ser487Ala. Consistent with a pathophysiological role for this modification, AMPKα1 Ser487 phosphorylation was inversely correlated with insulin sensitivity in human muscle. These data indicate a novel regulatory role of PKC to inhibit AMPKα1 in human cells. As PKC activation is associated with insulin resistance and obesity, PKC may underlie the reduced AMPK activity reported in response to overnutrition in insulin-resistant metabolic and vascular tissues. PMID:27784766

  12. Transphosphorylation of E. coli proteins during production of recombinant protein kinases provides a robust system to characterize kinase specificity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E.coli. We apply this to the study of the cytoplasmic domain of the plant rec...

  13. Dual activators of Protein Kinase R (PKR) and Protein Kinase R Like Kinase (PERK) Identify Common and Divergent Catalytic Targets

    PubMed Central

    Ming, Jie; Sun, Hong; Cao, Peng; Fusco, Dahlene N.; Chung, Raymond T.; Chorev, Michael; Jin, Qi; Aktas, Bertal H.

    2013-01-01

    Chemical genetics has evolved into a powerful tool for studying gene function in normal- and patho-biology. PKR and PERK, two eukaryotic translation initiation factor 2 alpha (eIF2α) kinases, play critical roles in maintenance of cellular hemostasis, metabolic stability, and anti-viral defenses. Both kinases interact with and phosphorylate additional substrates including tumor suppressor p53 and nuclear protein 90. Loss of function of both kinases has been studied by reverse genetics and recently identified inhibitors. In contrast, activating probes for studying the role of catalytic activity of these kinases are not available. We identified a 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5,7-dihydroxy-4H-chromen-4-one (DHBDC) as specific dual activator of PKR and PERK by screening a chemical library of 20,000 small molecules in a dual luciferase surrogate eIF2α phosphorylation assay. We present here extensive biological characterization and preliminary structure-activity relationship of DHBDC, which phosphorylate eIF2α by activating PKR and PERK but no other eIF2α kinases. These agents also activate downstream effectors of eIF2α phosphorylation; inducing CHOP and suppressing cyclin D1 expression and inhibiting cancer cell proliferation, all in a manner dependent on PKR and PERK. Consistent with the role of eIF2α phosphorylation in viral infection, DHBDC inhibits proliferation of human hepatitis C virus. Finally, DHBDC induces phosphorylation of Ikβα, and activates NF-κB pathway. Surprisingly, activation of NF-κB pathway is dependent on PERK but independent of PKR activity. These data indicate that DHBDC is an invaluable probe for elucidating the role of PKR and PERK in normal- and patho-biology. PMID:23784735

  14. Mitogen-activated protein kinases in male reproductive function

    PubMed Central

    Li, Michelle W.M.; Mruk, Dolores D.; Cheng, C. Yan

    2009-01-01

    Recent studies have shown that male reproductive function is modulated via the mitogen-activated protein kinase (MAPK) cascade. The MAPK cascade is involved in numerous male reproductive processes, including spermatogenesis, sperm maturation and activation, capacitation and acrosome reaction, before fertilization of the oocyte. In this review, we discuss the latest findings in this rapidly developing field regarding the role of MAPK in male reproduction in animal models and in human spermatozoa in vitro. This research will facilitate the design of future studies in humans, although much work is needed before this information can be used to manage male infertility and environmental toxicant-induced testicular injury in men, such as blood–testis-barrier disruption. PMID:19303360

  15. LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex

    PubMed Central

    Cirnaru, Maria D.; Marte, Antonella; Belluzzi, Elisa; Russo, Isabella; Gabrielli, Martina; Longo, Francesco; Arcuri, Ludovico; Murru, Luca; Bubacco, Luigi; Matteoli, Michela; Fedele, Ernesto; Sala, Carlo; Passafaro, Maria; Morari, Michele; Greggio, Elisa; Onofri, Franco; Piccoli, Giovanni

    2014-01-01

    Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains executing several functions, including GTP hydrolysis, kinase activity, and protein binding. Robust evidence suggests that LRRK2 acts at the synaptic site as a molecular hub connecting synaptic vesicles to cytoskeletal elements via a complex panel of protein-protein interactions. Here we investigated the impact of pharmacological inhibition of LRRK2 kinase activity on synaptic function. Acute treatment with LRRK2 inhibitors reduced the frequency of spontaneous currents, the rate of synaptic vesicle trafficking and the release of neurotransmitter from isolated synaptosomes. The investigation of complementary models lacking LRRK2 expression allowed us to exclude potential off-side effects of kinase inhibitors on synaptic functions. Next we studied whether kinase inhibition affects LRRK2 heterologous interactions. We found that the binding among LRRK2, presynaptic proteins and synaptic vesicles is affected by kinase inhibition. Our results suggest that LRRK2 kinase activity influences synaptic vesicle release via modulation of LRRK2 macro-molecular complex. PMID:24904275

  16. Resolution of thylakoid polyphenol oxidase and a protein kinase

    SciTech Connect

    Race, H.L.; Davenport, J.W.; Hind, G.

    1995-12-31

    The predominant protein kinase activity in octylglucoside (OG) extracts of spinach thylakoids has been attributed to a 64-kDa protein, tp64. Recent work calls into question the relation between tp64 and protein kinase activity, which were fractionated apart using fluid phase IEF and hydroxylapatite chromatography. Hind et al. sequenced tp64 from the cDNA and showed it to be a polyphenol oxidase (PPO) homolog. Its transit peptide indicates a location for the mature protein within the thylakoid lumen, where there is presumably no ATP and where it is remote from the presumed kinase substrates: the stromally exposed regions of integral PS-II membrane proteins. Here the authors suggest that the kinase is a 64-kDa protein distinct from tp64.

  17. The specificities of protein kinase inhibitors: an update.

    PubMed Central

    Bain, Jenny; McLauchlan, Hilary; Elliott, Matthew; Cohen, Philip

    2003-01-01

    We have previously examined the specificities of 28 commercially available compounds, reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases [Davies, Reddy, Caivano and Cohen (2000) Biochem. J. 351, 95-105]. In the present study, we have extended this analysis to a further 14 compounds. Of these, indirubin-3'-monoxime, SP 600125, KT 5823 and ML-9 were found to inhibit a number of protein kinases and conclusions drawn from their use in cell-based assays are likely to be erroneous. Kenpaullone, Alsterpaullone, Purvalanol, Roscovitine, pyrazolopyrimidine 1 (PP1), PP2 and ML-7 were more specific, but still inhibited two or more protein kinases with similar potency. Our results suggest that the combined use of Roscovitine and Kenpaullone may be useful for identifying substrates and physiological roles of cyclin-dependent protein kinases, whereas the combined use of Kenpaullone and LiCl may be useful for identifying substrates and physiological roles of glycogen synthase kinase 3. The combined use of SU 6656 and either PP1 or PP2 may be useful for identifying substrates of Src family members. Epigallocatechin 3-gallate, one of the main polyphenolic constituents of tea, inhibited two of the 28 protein kinases in the panel, dual-specificity, tyrosine-phosphorylated and regulated kinase 1A (DYRK1A; IC(50)=0.33 microM) and p38-regulated/activated kinase (PRAK; IC(50)=1.0 microM). PMID:12534346

  18. Auto-phosphorylation Represses Protein Kinase R Activity

    PubMed Central

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

    2017-01-01

    The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity. PMID:28281686

  19. Segmental helical motions and dynamical asymmetry modulate histidine kinase autophosphorylation.

    PubMed

    Mechaly, Ariel E; Sassoon, Nathalie; Betton, Jean-Michel; Alzari, Pedro M

    2014-01-01

    Histidine kinases (HKs) are dimeric receptors that participate in most adaptive responses to environmental changes in prokaryotes. Although it is well established that stimulus perception triggers autophosphorylation in many HKs, little is known on how the input signal propagates through the HAMP domain to control the transient interaction between the histidine-containing and ATP-binding domains during the catalytic reaction. Here we report crystal structures of the full cytoplasmic region of CpxA, a prototypical HK involved in Escherichia coli response to envelope stress. The structural ensemble, which includes the Michaelis complex, unveils HK activation as a highly dynamic process, in which HAMP modulates the segmental mobility of the central HK α-helices to promote a strong conformational and dynamical asymmetry that characterizes the kinase-active state. A mechanical model based on our structural and biochemical data provides insights into HAMP-mediated signal transduction, the autophosphorylation reaction mechanism, and the symmetry-dependent control of HK kinase/phosphatase functional states.

  20. Pr-specific phytochrome phosphorylation in vitro by a protein kinase present in anti-phytochrome maize immunoprecipitates

    NASA Technical Reports Server (NTRS)

    Biermann, B. J.; Pao, L. I.; Feldman, L. J.

    1994-01-01

    Protein kinase activity has repeatedly been found to co-purify with the plant photoreceptor phytochrome, suggesting that light signals received by phytochrome may be transduced or modulated through protein phosphorylation. In this study immunoprecipitation techniques were used to characterize protein kinase activity associated with phytochrome from maize (Zea mays L.). A protein kinase that specifically phosphorylated phytochrome was present in washed anti-phytochrome immunoprecipitates of etiolated coleoptile proteins. No other substrate tested was phosphorylated by this kinase. Adding salts or detergents to disrupt low-affinity protein interactions reduced background phosphorylation in immunoprecipitates without affecting phytochrome phosphorylation, indicating that the protein kinase catalytic activity is either intrinsic to the phytochrome molecule or associated with it by high-affinity interactions. Red irradiation (of coleoptiles or extracts) sufficient to approach photoconversion saturation reduced phosphorylation of immunoprecipitated phytochrome. Subsequent far-red irradiation reversed the red-light effect. Phytochrome phosphorylation was stimulated about 10-fold by a co-immunoprecipitated factor. The stimulatory factor was highest in immunoprecipitates when Mg2+ was present in immunoprecipitation reactions but remained in the supernatant in the absence of Mg2+. These observations provide strong support for the hypothesis that phytochrome-associated protein kinase modulates light responses in vivo. Since only phytochrome was found to be phosphorylated, the co-immunoprecipitated protein kinase may function to regulate receptor activity.

  1. Activation of fat cell adenylate cyclase by protein kinase C

    SciTech Connect

    Naghshineh, S.; Noguchi, M.; Huang, K.P.; Londos, C.

    1986-05-01

    Purified protein kinase C (C-kinase) from guinea pig pancreas and rat brain stimulated adenylate cyclase activity in purified rat adipocyte membranes. Cyclase stimulation occurred over 100 to 1000 mU/ml of C-kinase activity, required greater than 10 ..mu..M calcium, proceeded without a lag, was not readily reversible, and required no exogenous phospholipid. Moreover, C-kinase inhibitors, such as chlorpromazine and palmitoyl carnitine, inhibited selectively adenylate cyclase which was activated by C-kinase and calcium. Depending on assay conditions, 10 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) either enhanced or was required for kinase action on cyclase. Also, TPA plus calcium promoted the quantitative association of C-kinase with membranes. Adenylate cyclase activation by C-kinase was seen both in the presence and absence of exogenous GTP, indicating that the kinase effect does not result from an action on the GTP-binding, inhibitory regulatory component (N/sub i/) of the cyclase system. Moreover, the kinase effect was seen in the presence of non-phosphorylating ATP analogs, such as AppNHp and AppCH/sub 2/p, suggesting that the effects of C-kinase described herein may result from association with, rather than phosphorylation of, adenylate cyclase.

  2. Pomegranate fruit extract modulates UV-B-mediated phosphorylation of mitogen-activated protein kinases and activation of nuclear factor kappa B in normal human epidermal keratinocytes paragraph sign.

    PubMed

    Afaq, Farrukh; Malik, Arshi; Syed, Deeba; Maes, Daniel; Matsui, Mary S; Mukhtar, Hasan

    2005-01-01

    Excessive exposure of solar ultraviolet (UV) radiation, particularly its UV-B component, to humans causes many adverse effects that include erythema, hyperplasia, hyperpigmentation, immunosuppression, photoaging and skin cancer. In recent years, there is increasing use of botanical agents in skin care products. Pomegranate derived from the tree Punica granatum contains anthocyanins (such as delphinidin, cyanidin and pelargonidin) and hydrolyzable tannins (such as punicalin, pedunculagin, punicalagin, gallagic and ellagic acid esters of glucose) and possesses strong antioxidant and anti-inflammatory properties. Recently, we have shown that pomegranate fruit extract (PFE) possesses antitumor promoting effects in a mouse model of chemical carcinogenesis. To begin to establish the effect of PFE for humans in this study, we determined its effect on UV-B-induced adverse effects in normal human epidermal keratinocytes (NHEK). We first assessed the effect of PFE on UV-B-mediated phosphorylation of mitogen-activated protein kinases (MAPK) pathway in NHEK. Immunoblot analysis demonstrated that the treatment of NHEK with PFE (10-40 microg/mL) for 24 h before UV-B (40 mJ/cm(2)) exposure dose dependently inhibited UV-B-mediated phosphorylation of ERKl/2, JNK1/2 and p38 protein. We also observed that PFE (20 microg/mL) inhibited UV-B-mediated phosphorylation of MAPK in a time-dependent manner. Furthermore, in dose- and time-dependent studies, we evaluated the effect of PFE on UV-B-mediated activation of nuclear factor kappa B (NF-kappaB) pathway. Using Western blot analysis, we found that PFE treatment of NHEK resulted in a dose- and time-dependent inhibition of UV-B-mediated degradation and phosphorylation of IkappaBalpha and activation of IKKalpha. Using immunoblot analysis, enzyme-linked immunosorbent assay and electrophoretic mobility shift assay, we found that PFE treatment to NHEK resulted in a dose- and time-dependent inhibition of UV-B-mediated nuclear translocation and

  3. The Link between Protein Kinase CK2 and Atypical Kinase Rio1

    PubMed Central

    Kubiński, Konrad; Masłyk, Maciej

    2017-01-01

    The atypical kinase Rio1 is widespread in many organisms, ranging from Archaebacteria to humans, and is an essential factor in ribosome biogenesis. Little is known about the protein substrates of the enzyme and small-molecule inhibitors of the kinase. Protein kinase CK2 was the first interaction partner of Rio1, identified in yeast cells. The enzyme from various sources undergoes CK2-mediated phosphorylation at several sites and this modification regulates the activity of Rio1. The aim of this review is to present studies of the relationship between the two different kinases, with respect to CK2-mediated phosphorylation of Rio1, regulation of Rio1 activity, and similar susceptibility of the kinases to benzimidazole inhibitors. PMID:28178206

  4. Subcutaneous administration of liraglutide ameliorates learning and memory impairment by modulating tau hyperphosphorylation via the glycogen synthase kinase-3β pathway in an amyloid β protein induced alzheimer disease mouse model.

    PubMed

    Qi, Liqin; Ke, Linfang; Liu, Xiaohong; Liao, Lianming; Ke, Sujie; Liu, Xiaoying; Wang, Yanping; Lin, Xiaowei; Zhou, Yu; Wu, Lijuan; Chen, Zhou; Liu, Libin

    2016-07-15

    Type 2 diabetes mellitus is a risk factor for Alzheimer's disease (AD). The glucagon-like peptide-1 analog liraglutide, a novel long-lasting incretin hormone, has been used to treat type 2 diabetes mellitus. In addition, liraglutide has been shown to be neurotrophic and neuroprotective. Here, we investigated the effects of liraglutide on amyloid β protein (Aβ)-induced AD in mice and explored its mechanism of action. The results showed that subcutaneous administration of liraglutide (25nmol/day), once daily for 8 weeks, prevented memory impairments in the Y Maze and Morris Water Maze following Aβ1-42 intracerebroventricular injection, and alleviated the ultra-structural changes of pyramidal neurons and chemical synapses in the hippocampal CA1 region. Furthermore, liraglutide reduced Aβ1-42-induced tau phosphorylation via the protein kinase B and glycogen synthase kinase-3β pathways. Thus liraglutide may alleviate cognitive impairment in AD by at least decreasing the phosphorylation of tau.

  5. A family of human cdc2-related protein kinases.

    PubMed Central

    Meyerson, M; Enders, G H; Wu, C L; Su, L K; Gorka, C; Nelson, C; Harlow, E; Tsai, L H

    1992-01-01

    The p34cdc2 protein kinase is known to regulate important transitions in the eukaryotic cell cycle. We have identified 10 human protein kinases based on their structural relation to p34cdc2. Seven of these kinases are novel and the products of five share greater than 50% amino acid sequence identity with p34cdc2. The seven novel genes are broadly expressed in human cell lines and tissues with each displaying some cell type or tissue specificity. The cdk3 gene, like cdc2 and cdk2, can complement cdc28 mutants of Saccharomyces cerevisiae, suggesting that all three of these protein kinases can play roles in the regulation of the mammalian cell cycle. The identification of a large family of cdc2-related kinases opens the possibility of combinatorial regulation of the cell cycle together with the emerging large family of cyclins. Images PMID:1639063

  6. The protein activator of protein kinase R, PACT/RAX, negatively regulates protein kinase R during mouse anterior pituitary development.

    PubMed

    Dickerman, Benjamin K; White, Christine L; Kessler, Patricia M; Sadler, Anthony J; Williams, Bryan R G; Sen, Ganes C

    2015-12-01

    The murine double-stranded RNA-binding protein termed protein kinase R (PKR)-associated protein X (RAX) and the human homolog, protein activator of PKR (PACT), were originally characterized as activators of PKR. Mice deficient in RAX show reproductive and developmental defects, including reduced body size, craniofacial defects and anterior pituitary hypoplasia. As these defects are not observed in PKR-deficient mice, the phenotype has been attributed to PKR-independent activities of RAX. Here we further investigated the involvement of PKR in the physiological function of RAX, by generating rax(-/-) mice deficient in PKR, or carrying a kinase-inactive mutant of PKR (K271R) or an unphosphorylatable mutant of the PKR substrate eukaryotic translation initiation factor 2 α subunit (eIF2α) (S51A). Ablating PKR expression rescued the developmental and reproductive deficiencies in rax(-/-) mice. Generating rax(-/-) mice with a kinase-inactive mutant of PKR resulted in similar rescue, confirming that the rax(-/-) defects are PKR dependent; specifically that the kinase activity of PKR was required for these defects. Moreover, generating rax(-/-) mice that were heterozygous for an unphosphorylatable mutant eIF2α provides partial rescue of the rax(-/-) defect, consistent with mutation of one copy of the Eif2s1 gene. These observations were further investigated in vitro by reducing RAX expression in anterior pituitary cells, resulting in increased PKR activity and induction of the PKR-regulated cyclin-dependent kinase inhibitor p21(WAF1/CIP1). These results demonstrate that PKR kinase activity is required for onset of the rax(-/-) phenotype, implying an unexpected function for RAX as a negative regulator of PKR in the context of postnatal anterior pituitary tissue, and identify a critical role for the regulation of PKR activity for normal development.

  7. EBNA3C-mediated regulation of aurora kinase B contributes to Epstein-Barr virus-induced B-cell proliferation through modulation of the activities of the retinoblastoma protein and apoptotic caspases.

    PubMed

    Jha, Hem Chandra; Lu, Jie; Saha, Abhik; Cai, Qiliang; Banerjee, Shuvomoy; Prasad, Mahadesh A J; Robertson, Erle S

    2013-11-01

    Epstein-Barr virus (EBV) is an oncogenic gammaherpesvirus that is implicated in several human malignancies, including Burkitt's lymphoma (BL), posttransplant lymphoproliferative disease (PTLD), nasopharyngeal carcinoma (NPC), and AIDS-associated lymphomas. Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential EBV latent antigens, can induce mammalian cell cycle progression through its interaction with cell cycle regulators. Aurora kinase B (AK-B) is important for cell division, and deregulation of AK-B is associated with aneuploidy, incomplete mitotic exit, and cell death. Our present study shows that EBNA3C contributes to upregulation of AK-B transcript levels by enhancing the activity of its promoter. Further, EBNA3C also increased the stability of the AK-B protein, and the presence of EBNA3C leads to reduced ubiquitination of AK-B. Importantly, EBNA3C in association with wild-type AK-B but not with its kinase-dead mutant led to enhanced cell proliferation, and AK-B knockdown can induce nuclear blebbing and cell death. This phenomenon was rescued in the presence of EBNA3C. Knockdown of AK-B resulted in activation of caspase 3 and caspase 9, along with poly(ADP-ribose) polymerase 1 (PARP1) cleavage, which is known to be an important contributor to apoptotic signaling. Importantly, EBNA3C failed to stabilize the kinase-dead mutant of AK-B compared to wild-type AK-B, which suggests a role for the kinase domain in AK-B stabilization and downstream phosphorylation of the cell cycle regulator retinoblastoma protein (Rb). This study demonstrates the functional relevance of AK-B kinase activity in EBNA3C-regulated B-cell proliferation and apoptosis.

  8. RAF protein-serine/threonine kinases: Structure and regulation

    SciTech Connect

    Roskoski, Robert

    2010-08-27

    Research highlights: {yields} The formation of unique side-to-side RAF dimers is required for full kinase activity. {yields} RAF kinase inhibitors block MEK activation in cells containing oncogenic B-RAF. {yields} RAF kinase inhibitors can lead to the paradoxical increase in RAF kinase activity. -- Abstract: A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.

  9. p38β Mitogen-Activated Protein Kinase Modulates Its Own Basal Activity by Autophosphorylation of the Activating Residue Thr180 and the Inhibitory Residues Thr241 and Ser261

    PubMed Central

    Beenstock, Jonah; Melamed, Dganit; Mooshayef, Navit; Mordechay, Dafna; Garfinkel, Benjamin P.; Ahn, Natalie G.; Admon, Arie

    2016-01-01

    Many enzymes are self-regulated and can either inhibit or enhance their own catalytic activity. Enzymes that do both are extremely rare. Many protein kinases autoactivate by autophosphorylating specific sites at their activation loop and are inactivated by phosphatases. Although mitogen-activated protein kinases (MAPKs) are usually activated by dual phosphorylation catalyzed by MAPK kinases (MAPKKs), the MAPK p38β is exceptional and is capable of self-activation by cis autophosphorylation of its activation loop residue T180. We discovered that p38β also autophosphorylates in trans two previously unknown sites residing within a MAPK-specific structural element known as the MAPK insert: T241 and S261. Whereas phosphorylation of T180 evokes catalytic activity, phosphorylation of S261 reduces the activity of T180-phosphorylated p38β, and phosphorylation of T241 reduces its autophosphorylation in trans. Both phosphorylations do not affect the activity of dually phosphorylated p38β. T241 of p38β is found phosphorylated in vivo in bone and muscle tissues. In myogenic cell lines, phosphorylation of p38β residue T241 is correlated with differentiation to myotubes. T241 and S261 are also autophosphorylated in intrinsically active variants of p38α, but in this protein, they probably play a different role. We conclude that p38β is an unusual enzyme that automodulates its basal, MAPKK-independent activity by several autophosphorylation events, which enhance and suppress its catalytic activity. PMID:26976637

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

    NASA Technical Reports Server (NTRS)

    Li, H.; Roux, S. J.

    1992-01-01

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

  11. Reciprocal Regulation of Aquaporin-2 Abundance and Degradation by Protein Kinase A and p38-MAP Kinase

    PubMed Central

    Nedvetsky, Pavel I.; Tabor, Vedrana; Tamma, Grazia; Beulshausen, Sven; Skroblin, Philipp; Kirschner, Aline; Mutig, Kerim; Boltzen, Mareike; Petrucci, Oscar; Vossenkämper, Anna; Wiesner, Burkhard; Bachmann, Sebastian; Rosenthal, Walter

    2010-01-01

    Arginine-vasopressin (AVP) modulates the water channel aquaporin-2 (AQP2) in the renal collecting duct to maintain homeostasis of body water. AVP binds to vasopressin V2 receptors (V2R), increasing cAMP, which promotes the redistribution of AQP2 from intracellular vesicles into the plasma membrane. cAMP also increases AQP2 transcription, but whether altered degradation also modulates AQP2 protein levels is not well understood. Here, elevation of cAMP increased AQP2 protein levels within 30 minutes in primary inner medullary collecting duct (IMCD) cells, in human embryonic kidney (HEK) 293 cells ectopically expressing AQP2, and in mouse kidneys. Accelerated transcription or translation did not explain this increase in AQP2 abundance. In IMCD cells, cAMP inhibited p38-mitogen-activated protein kinase (p38-MAPK) via activation of protein kinase A (PKA). Inhibition of p38-MAPK associated with decreased phosphorylation (serine 261) and polyubiquitination of AQP2, preventing proteasomal degradation. Our results demonstrate that AVP enhances AQP2 protein abundance by altering its proteasomal degradation through a PKA- and p38-MAPK–dependent pathway. PMID:20724536

  12. THI1, a Thiamine Thiazole Synthase, Interacts with Ca2+-Dependent Protein Kinase CPK33 and Modulates the S-Type Anion Channels and Stomatal Closure in Arabidopsis1[OPEN

    PubMed Central

    Li, Chun-Long; Wang, Mei; Wu, Xiao-Meng; Chen, Dong-Hua; Lv, Hong-Jun; Shen, Jian-Lin; Qiao, Zhu; Zhang, Wei

    2016-01-01

    Thiamine is required for both plant growth and development. Here, the involvement of a thiamine thiazole synthase, THI1, has been demonstrated in both guard cell abscisic acid (ABA) signaling and the drought response in Arabidopsis (Arabidopsis thaliana). THI1 overexpressors proved to be more sensitive to ABA than the wild type with respect to both the activation of guard cell slow type anion channels and stomatal closure; this effectively reduced the rate of water loss from the plant and thereby enhanced its level of drought tolerance. A yeast two-hybrid strategy was used to screen a cDNA library from epidermal strips of leaves for THI1 regulatory factors, and identified CPK33, a Ca2+-dependent protein kinase, as interactor with THI1 in a plasma membrane-delimited manner. Loss-of-function cpk33 mutants were hypersensitive to ABA activation of slow type anion channels and ABA-induced stomatal closure, while the CPK33 overexpression lines showed opposite phenotypes. CPK33 kinase activity was essential for ABA-induced stomatal closure. Consistent with their contrasting regulatory role over stomatal closure, THI1 suppressed CPK33 kinase activity in vitro. Together, our data reveal a novel regulatory role of thiamine thiazole synthase to kinase activity in guard cell signaling. PMID:26662273

  13. Interaction of SNF1 Protein Kinase with Its Activating Kinase Sak1▿

    PubMed Central

    Liu, Yang; Xu, Xinjing; Carlson, Marian

    2011-01-01

    The Saccharomyces cerevisiae SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change. PMID:21216941

  14. Protein kinase C activators inhibit capillary endothelial cell growth

    SciTech Connect

    Doctrow, S.R.

    1986-05-01

    Phorbol 12,13-dibutyrate (PDBu) binds specifically to bovine capillary endothelial (BCE) cells (K/sub d/ = 8nM) and inhibits the proliferation (K/sub 50/ = 6 +/- 4 nM). Under similar conditions, PDBu does not inhibit the growth of bovine aortic endothelial or smooth muscle cells. PDBu markedly attenuates the response of BCE cells to purified human hepatoma-derived growth factor which, in the absence of PDBu, stimulates BCE cell growth by about 3-fold. Several observations suggest that the inhibition of BCE cell growth by PDBu is mediated by protein kinase C: (1) different phorbol compounds inhibit BCE cell growth according to the relative potencies as protein kinase C activators (12-tetradecanoylphorbol 13-acetate > PDBu >> phorbol 12,13-diacetate >>>..beta..-phorbol; ..cap alpha..-phorbol 12,13-didecanoate). (2) Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol (diC/sub 8/), a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2-dibutyrylglycerol, does not affect PDBu binding. (3) A cytosolic extract from BCE cells contains a Ca/sup 2 +//phosphatidylserine-dependent kinase that is activated by diC/sub 8/ and PDBu, but not by ..beta..-phorbol. These results support a role for protein kinase C in suppressing capillary endothelial cell growth and may therefore have implications in the intracellular regulation of angiogenesis.

  15. Characterization of Protein Kinase CK2 from Trypanosoma brucei

    PubMed Central

    Jensen, Bryan C; Kifer, Charles T; Brekken, Deirdre L; Randall, Amber C; Wang, Qin; Drees, Becky L.; Parsons, Marilyn

    2007-01-01

    CK2 is a ubiquitous but enigmatic kinase. The difficulty in assigning a role to CK2 centers on the fact that, to date, no biologically relevant modulator of its function has been identified. One common theme revolves around a constellation of known substrates involved in growth control, compatible with its concentration in the nucleus and nucleolus. We had previously described the identification of two catalytic subunits of CK2 in Trypanosoma brucei and characterized one of them. Here we report the characterization of the second catalytic subunit, CK2α’, and the identification and characterization of the regulatory subunit CK2β. All three subunits are primarily localized to the nucleolus in T. brucei. We also show that CK2β interacts with the nucleolar protein NOG1, adding to the interaction map which previously linked CK2α to the nucleolar protein NOPP44/46, which in turn associates with the rRNA binding protein p37. CK2 activity has four distinctive features: near equal affinity for GTP and ATP, heparin sensitivity, and stimulation by polyamines and polybasic peptides. Sequence comparison shows that the parasite orthologues have mutations in residues previously mapped as important in specifying affinity for GTP and stimulation by both polyamines and polybasic peptides. Studies of the enzymatic activity of the T. brucei CK2s show that both the affinity for GTP and stimulation by polyamines have been lost and only the features of heparin inhibition and stimulation by polybasic peptides are conserved. PMID:17097160

  16. Characterization of microtubule-associated protein 1-associated protein kinases from rat brain.

    PubMed

    Fujii, T; Watanabe, M; Nakamura, A

    1996-01-01

    The microtubule-associated protein (MAP) 1 preparation, MAP1A and 1B, obtained from rat brain microtubules was associated with protein kinases that were insensitive to cAMP, cGMP, calcium, calcium/calmodulin and calcium/phosphatidylserine. The fractionation of highly purified MAP1 by phosphocellulose chromatography revealed that protein kinase activity to phosphorylate phosvitin was separated into three major peaks (MAP1 kinases A, B and C). MAP1 was recovered in the MAP1 kinase A fraction and phosphorylated by the contained kinase. MAP1 kinase A is a novel protein kinase that is remarkably activated by poly-L-lysine and poly-L-arginine, but very insensitive to heparin among the kinases. Photoaffinity labeling using [alpha-32P]8-azido ATP indicated that the 65 kDa polypeptide is identified as an ATP-binding protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the highly purified MAP1 and MAP1 kinase A fractions. MAP1 kinases B and C may be identified as casein kinase I- and II-like kinases. The present results show that MAP1 is associated with at least three kinases and provide an insight for understanding thoroughly the MAP1-mediated microtubule functions.

  17. Regulatory Crosstalk by Protein Kinases on CFTR Trafficking and Activity

    PubMed Central

    Farinha, Carlos M.; Swiatecka-Urban, Agnieszka; Brautigan, David L.; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease. PMID:26835446

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

  19. A tumor suppressor C53 protein antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation

    PubMed Central

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

    2009-01-01

    Cyclin dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint (1). More recently, Wang et al (2007) found that C53/LZAP may function as a tumor suppressor via inhibiting NF-κB signaling (2). We report here identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexrepsssion. Intriguingly, we found that C53 interacts with checkpoint kinase 1 (Chk1) and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell cycle progression and DNA damage response. PMID:19223857

  20. The Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway as a Discovery Target in Stroke.

    PubMed

    Sun, Jing; Nan, Guangxian

    2016-05-01

    Protein kinases are critical modulators of a variety of intracellular and extracellular signal transduction pathways, and abnormal phosphorylation events can contribute to disease progression in a variety of diseases. As a result, protein kinases have emerged as important new drug targets for small molecule therapeutics. The mitogen-activated protein kinase (MAPK) signaling pathway transmits signals from the cell membrane to the nucleus in response to a variety of different stimuli. Because this pathway controls a broad spectrum of cellular processes, including growth, inflammation, and stress responses, it is accepted as a therapeutic target for cancer and peripheral inflammatory disorders. There is also increasing evidence that MAPK is an important regulator of ischemic and hemorrhagic cerebral vascular disease, raising the possibility that it might be a drug discovery target for stroke. In this review, we discuss the MAPK signaling pathway in association with its activation in stroke-induced brain injury.

  1. Regulated protein kinases and phosphatases in cell cycle decisions.

    PubMed

    Novak, Bela; Kapuy, Orsolya; Domingo-Sananes, Maria Rosa; Tyson, John J

    2010-12-01

    Many aspects of cell physiology are controlled by protein kinases and phosphatases, which together determine the phosphorylation state of targeted substrates. Some of these target proteins are themselves kinases or phosphatases or other components of a regulatory network characterized by feedback and feed-forward loops. In this review we describe some common regulatory motifs involving kinases, phosphatases, and their substrates, focusing particularly on bistable switches involved in cellular decision processes. These general principles are applied to cell cycle transitions, with special emphasis on the roles of regulated phosphatases in orchestrating progression from one phase to the next of the DNA replication-division cycle.

  2. The C-terminal tail of protein kinase D2 and protein kinase D3 regulates their intracellular distribution

    SciTech Connect

    Papazyan, Romeo; Rozengurt, Enrique; Rey, Osvaldo . E-mail: orey@mednet.ucla.edu

    2006-04-14

    We generated a set of GFP-tagged chimeras between protein kinase D2 (PKD2) and protein kinase D3 (PKD3) to examine in live cells the contribution of their C-terminal region to their intracellular localization. We found that the catalytic domain of PKD2 and PKD3 can localize to the nucleus when expressed without other kinase domains. However, when the C-terminal tail of PKD2 was added to its catalytic domain, the nuclear localization of the resulting protein was inhibited. In contrast, the nuclear localization of the CD of PKD3 was not inhibited by its C-terminal tail. Furthermore, the exchange of the C-terminal tail of PKD2 and PKD3 in the full-length proteins was sufficient to exchange their intracellular localization. Collectively, these data demonstrate that the short C-terminal tail of these kinases plays a critical role in determining their cytoplasmic/nuclear localization.

  3. Targeting protein kinases in central nervous system disorders

    PubMed Central

    Chico, Laura K.; Van Eldik, Linda J.; Watterson, D. Martin

    2010-01-01

    Protein kinases are a growing drug target class in disorders in peripheral tissues, but the development of kinase-targeted therapies for central nervous system (CNS) diseases remains a challenge, largely owing to issues associated specifically with CNS drug discovery. However, several candidate therapeutics that target CNS protein kinases are now in various stages of preclinical and clinical development. We review candidate compounds and discuss selected CNS protein kinases that are emerging as important therapeutic targets. In addition, we analyse trends in small-molecule properties that correlate with key challenges in CNS drug discovery, such as blood–brain barrier penetrance and cytochrome P450-mediated metabolism, and discuss the potential of future approaches that will integrate molecular-fragment expansion with pharmacoinformatics to address these challenges. PMID:19876042

  4. Protein Kinases and Parkinson’s Disease

    PubMed Central

    Mehdi, Syed Jafar; Rosas-Hernandez, Hector; Cuevas, Elvis; Lantz, Susan M.; Barger, Steven W.; Sarkar, Sumit; Paule, Merle G.; Ali, Syed F.; Imam, Syed Z.

    2016-01-01

    Currently, the lack of new drug candidates for the treatment of major neurological disorders such as Parkinson’s disease has intensified the search for drugs that can be repurposed or repositioned for such treatment. Typically, the search focuses on drugs that have been approved and are used clinically for other indications. Kinase inhibitors represent a family of popular molecules for the treatment and prevention of various cancers, and have emerged as strong candidates for such repurposing because numerous serine/threonine and tyrosine kinases have been implicated in the pathobiology of Parkinson’s disease. This review focuses on various kinase-dependent pathways associated with the expression of Parkinson’s disease pathology, and evaluates how inhibitors of these pathways might play a major role as effective therapeutic molecules. PMID:27657053

  5. Phosphorylation of the Kinase Interaction Motif in Mitogen-activated Protein (MAP) Kinase Phosphatase-4 Mediates Cross-talk between Protein Kinase A and MAP Kinase Signaling Pathways*

    PubMed Central

    Dickinson, Robin J.; Delavaine, Laurent; Cejudo-Marín, Rocío; Stewart, Graeme; Staples, Christopher J.; Didmon, Mark P.; Trinidad, Antonio Garcia; Alonso, Andrés; Pulido, Rafael; Keyse, Stephen M.

    2011-01-01

    MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site 55RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo. PMID:21908610

  6. The Final Link: Tapping the Power of Chemical Genetics to Connect the Molecular and Biologic Functions of Mitotic Protein Kinases

    PubMed Central

    Lera, Robert F.; Burkard, Mark E.

    2013-01-01

    During mitosis, protein kinases coordinate cellular reorganization and chromosome segregation to ensure accurate distribution of genetic information into daughter cells. Multiple protein kinases contribute to mitotic regulation, modulating molecular signaling more rapidly than possible with gene expression. However, a comprehensive understanding of how kinases regulate mitotic progression remains elusive. The challenge arises from multiple functions and substrates, a large number of “bystander” phosphorylation events, and the brief window in which all mitotic events transpire. Analog-sensitive alleles of protein kinases are powerful chemical genetic tools for rapid and specific interrogation of kinase function. Moreover, combining these tools with advanced proteomics and substrate labeling has identified phosphorylation sites on numerous protein targets. Here, we review the chemical genetic tools available to study kinase function and identify substrates. We describe how chemical genetics can also be used to link kinase function with cognate phosphorylation events to provide mechanistic detail. This can be accomplished by dissecting subsets of kinase functions and chemical genetic complementation. We believe a complete “chemical genetic toolbox” will ultimately allow a comprehensive understanding of how protein kinases regulate mitosis. PMID:23075814

  7. Inhibition of epithelial Na sup + transport by atriopeptin, protein kinase c, and pertussis toxin

    SciTech Connect

    Mohrmann, M.; Cantiello, H.F.; Ausiello, D.A. )

    1987-08-01

    The authors have recently shown the selective inhibition of an amiloride-sensitive, conductive pathway for Na{sup +} by atrial natriuretic peptide and 8-bromoguanosine 3{prime},5{prime}-cyclic monophosphate (8-BrcGMP) in the renal epithelial cell line, LLC-PK{sub i}. Using {sup 22}Na{sup +} fluxes, they further investigated the modulation of Na{sup +} transport by atrial natriuretic peptide and by agents that increase cGMP production, activate protein kinase c, or modulate guanine nucleotide regulatory protein function. Sodium nitroprusside increases intracellular cGMP concentrations without affecting cAMP concentrations and completely inhibits amiloride-sensitive Na{sup +} uptake in a time- and concentration-dependent manner. Oleoyl 2-acetylglycerol and phorbol 12-myristate 13-acetate, activators of protein kinase c, inhibit Na{sup +} uptake by 93 {plus minus} 13 and 51 {plus minus} 10%, respectively. Prolonged incubation with phorbol ester results in the downregulation of protein kinase c activity and reduces the inhibitory effect of atrial natriuretic peptide, suggesting that the action of this peptide involves stimulation of protein kinase c. Pertussis toxin, which induces the ADP-ribosylation of a 41-kDa guanine nucleotide regulatory protein in LLC-PK{sub i} cells, inhibits {sup 22}Na{sup +} influx to the same extent as amiloride. Thus, increasing cGMP, activating protein kinase c, and ADP-ribosylating a guanine nucleotide regulatory protein all inhibit Na{sup +} uptake. These events may be sequentially involved in the action of atrial natriuretic peptide.

  8. Development of Novel In Vivo Chemical Probes to Address CNS Protein Kinase Involvement in Synaptic Dysfunction

    PubMed Central

    Watterson, D. Martin; Grum-Tokars, Valerie L.; Roy, Saktimayee M.; Schavocky, James P.; Bradaric, Brinda Desai; Bachstetter, Adam D.; Xing, Bin; Dimayuga, Edgardo; Saeed, Faisal; Zhang, Hong; Staniszewski, Agnieszka; Pelletier, Jeffrey C.; Minasov, George; Anderson, Wayne F.; Arancio, Ottavio; Van Eldik, Linda J.

    2013-01-01

    Serine-threonine protein kinases are critical to CNS function, yet there is a dearth of highly selective, CNS-active kinase inhibitors for in vivo investigations. Further, prevailing assumptions raise concerns about whether single kinase inhibitors can show in vivo efficacy for CNS pathologies, and debates over viable approaches to the development of safe and efficacious kinase inhibitors are unsettled. It is critical, therefore, that these scientific challenges be addressed in order to test hypotheses about protein kinases in neuropathology progression and the potential for in vivo modulation of their catalytic activity. Identification of molecular targets whose in vivo modulation can attenuate synaptic dysfunction would provide a foundation for future disease-modifying therapeutic development as well as insight into cellular mechanisms. Clinical and preclinical studies suggest a critical link between synaptic dysfunction in neurodegenerative disorders and the activation of p38αMAPK mediated signaling cascades. Activation in both neurons and glia also offers the unusual potential to generate enhanced responses through targeting a single kinase in two distinct cell types involved in pathology progression. However, target validation has been limited by lack of highly selective inhibitors amenable to in vivo use in the CNS. Therefore, we employed high-resolution co-crystallography and pharmacoinformatics to design and develop a novel synthetic, active site targeted, CNS-active, p38αMAPK inhibitor (MW108). Selectivity was demonstrated by large-scale kinome screens, functional GPCR agonist and antagonist analyses of off-target potential, and evaluation of cellular target engagement. In vitro and in vivo assays demonstrated that MW108 ameliorates beta-amyloid induced synaptic and cognitive dysfunction. A serendipitous discovery during co-crystallographic analyses revised prevailing models about active site targeting of inhibitors, providing insights that will

  9. PROTEIN KINASE B/AKT IS A NOVEL CYSTEINE STRING PROTEIN KINASE THAT REGULATES EXOCYTOSIS RELEASE KINETICS AND QUANTAL SIZE

    PubMed Central

    Evans, Gareth J. O.; Barclay, Jeff W.; Prescott, Gerald R.; Jo, Sung-Ro; Burgoyne, Robert D.; Birnbaum, Morris J.; Morgan, Alan

    2008-01-01

    Protein kinase B/Akt has been implicated in the insulin-dependent exocytosis of GLUT4-containing vesicles, and, more recently, insulin secretion. To determine if Akt also regulates insulin-independent exocytosis, we used adrenal chromaffin cells, a popular neuronal model. Akt1 was the predominant isoform expressed in chromaffin cells, although lower levels of Akt2 and Akt3 were also found. Secretory stimuli in both intact and permeabilized cells induced Akt phosphorylation on serine-473, and the time course of Ca2+-induced Akt phosphorylation was similar to that of exocytosis in permeabilized cells. To determine if Akt modulated exocytosis, we transfected chromaffin cells with Akt constructs and monitored catecholamine release by amperometry. Wild-type Akt had no effect on the overall number of exocytotic events, but slowed the kinetics of catecholamine release from individual vesicles, resulting in an increased quantal size. This effect was due to phosphorylation by Akt, as it was not seen in cells transfected with kinase-dead mutant Akt. As overexpression of cysteine string protein (CSP) results in a similar alteration in release kinetics and quantal size, we determined if CSP was an Akt substrate. In vitro 32P-phosphorylation studies revealed that Akt phosphorylates CSP on serine-10. Using phospho-serine10-specific antisera, we found that both transfected and endogenous cellular CSP is phosphorylated by Akt on this residue. Taken together, these findings reveal a novel role for Akt phosphorylation in regulating the late stages of exocytosis and suggest that this is achieved via the phosphorylation of CSP on serine-10. PMID:16243840

  10. Mitochondria: a kinase anchoring protein 1, a signaling platform for mitochondrial form and function.

    PubMed

    Merrill, Ronald A; Strack, Stefan

    2014-03-01

    Mitochondria are best known for their role as cellular power plants, but they also serve as signaling hubs, regulating cellular proliferation, differentiation, and survival. A kinase anchoring protein 1 (AKAP1) is a scaffold protein that recruits protein kinase A (PKA) and other signaling proteins, as well as RNA, to the outer mitochondrial membrane. AKAP1 thereby integrates several second messenger cascades to modulate mitochondrial function and associated physiological and pathophysiological outcomes. Here, we review what is currently known about AKAP1's macromolecular interactions in health and disease states, including obesity. We also discuss dynamin-related protein 1 (Drp1), the enzyme that catalyzes mitochondrial fission, as one of the key substrates of the PKA/AKAP1 signaling complex in neurons. Recent evidence suggests that AKAP1 has critical roles in neuronal development and survival, which are mediated by inhibitory phosphorylation of Drp1 and maintenance of mitochondrial integrity.

  11. Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-epsilon.

    PubMed Central

    Schaap, D; van der Wal, J; van Blitterswijk, W J; van der Bend, R L; Ploegh, H L

    1993-01-01

    In signal transduction, diacylglycerol (DG) kinase attenuates levels of the second messenger DG by converting it to phosphatidic acid. A previously cloned full-length human 86 kDa DG kinase cDNA was expressed as fusion protein in Escherichia coli, to aid in the generation of DG-kinase-specific monoclonal antibodies suitable for immunoprecipitation experiments. To investigate whether phosphorylation of DG kinase is a possible mechanism for its regulation, COS-7 cells were transiently transfected with the DG kinase cDNA and phosphorylation of the expressed DG kinase was induced by various stimuli. Activation of both cyclic AMP-dependent protein kinase and protein kinase C (PKC) resulted in phosphorylation of DG kinase on serine residues in vivo, and both kinases induced this phosphorylation within the same tryptic phosphopeptide, suggesting that they may exert similar control over DG kinase. No phosphorylation was observed upon ionomycin treatment, intended to activate Ca2+/calmodulin-dependent kinases. Co-transfections of DG kinase with either PKC-alpha or PKC-epsilon cDNA revealed that both protein kinases, when stimulated, are able to phosphorylate DG kinase. For PKC-epsilon, DG kinase is the first in vivo substrate identified. Stimulation with epidermal growth factor (EGF) of COS-7 cells transfected with both DG kinase and EGF-receptor cDNA results mainly in phosphorylation of DG kinase on tyrosine. Since the EGF receptor has an intrinsic tyrosine kinase activity, this finding implies that DG kinase may be a direct substrate for the activated EGF receptor. Images Figure 2 Figure 3 Figure 4 PMID:7679574

  12. An unusual receptor tyrosine kinase of Schistosoma mansoni contains a Venus Flytrap module.

    PubMed

    Vicogne, Jérôme; Pin, Jean Philippe; Lardans, Vinca; Capron, Monique; Noël, Christophe; Dissous, Colette

    2003-01-01

    Previous studies have suggested that successful development of the parasitic helminth Schistosoma mansoni must be dependent on an adaptative molecular dialogue with its hosts and on the existence of receptors for growth factors and hormones. Attempts to identify a homolog of the insulin receptor (IR) have led us to characterize a new receptor tyrosine kinase (RTK) molecule in S. mansoni. SmRTK-1 is an integral membrane protein with a single membrane-spanning sequence separating an extracellular ligand-binding domain and a cytoplasmic TK domain. Structural and phylogenetic analyses of the kinase domain of SmRTK-1 confirmed its similarity to IR catalytic domains. However, sequence analysis of the extracellular domain of SmRTK-1 revealed similarity with various proteins (such as drug receptors) that share a structure known as the Venus Flytrap (VFT) module. Alignment with other VFT modules for which the structure has been solved was used to generate a 3D model of the putative VFT module of SmRTK-1. Phylogenetic analysis indicated that the SmRTK-1 VFT module was closer to that of the GABA(B) receptor. Numerous RTK genes recently discovered in vertebrate and invertebrate species code for large families of modular proteins with diverse structures and ligand-binding specificities. SmRTK-1 probably represents a new class of RTK whose function remains to be determined. RTKs are present in all metazoans and associated with the control of metabolism, growth and development. The preferential localization of SmRTK-1 in sporocyst germinal cells and ovocytes could be in favor of its function in schistosome growth and differentiation.

  13. Partial purification of a spinach thylakoid protein kinase that can phosphorylate light-harvesting chlorophyll a/b proteins

    SciTech Connect

    Clark, R.D.; Hind, G.; Bennett, J.

    1985-01-01

    Protein phosphorylation in plant tissues is particularly marked in chloroplasts, protein kinase activity being associated with the outer envelope, the soluble stromal fraction, and the thylakoid membrane. Furthermore, thylakoid-bound activity probably includes several distinct kinases, as suggested by studies of divalent cation specificity and thermal lability carried out with intact thylakoids and by subfractionation of solubilized membranes. Illumination of thylakoids, particularly with red light, promotes the rapid and extensive phosphorylation of the light-harvesting chlorophyll a/b complex (LHCII) on a threonine residue near the amino terminus of the protein. This phosphorylation is thought to be involved in regulating the distribution of absorbed quanta between photosystems II and I and is modulated by the redox state of the thylakoid plastoquinone pool. Neither of the thylakoid kinases reported to date was capable of phosphorylating purified LHCII in vitro or of incorporating phosphate into threonyl residues of exogenous substrates, that some LHCII phosphorylation was catalyzed by a preliminary fraction led workers to suggest that at least one other kinase remained to be isolated. Here, the authors report the solubilization and partial purification of a protein kinase from spinach thylakoids that is capable of phosphorylating LHCII in vitro, and they show that the specific site of phosphorylation is very nearly the same as, if not identical with, the site phosphorylated in organello.

  14. Protein kinase CK2 in development and differentiation

    PubMed Central

    Götz, Claudia; Montenarh, Mathias

    2017-01-01

    Among the human kinomes, protein kinase CK2 (formerly termed casein kinase II) is considered to be essential, as it is implicated in the regulation of various cellular processes. Experiments with pharmacological inhibitors of the kinase activity of CK2 provide evidence that CK2 is essential for development and differentiation. Therefore, the present review addresses the role of CK2 during embryogenesis, neuronal, adipogenic, osteogenic and myogenic differentiation in established model cell lines, and in embryonic, neural and mesenchymal stem cells. CK2 kinase activity appears to be essential in the early stages of differentiation, as CK2 inhibition at early time points generally prevents differentiation. In addition, the present review reports on target proteins of CK2 in embryogenesis and differentiation. PMID:28357063

  15. Allosteric activation of apicomplexan calcium-dependent protein kinases

    PubMed Central

    Ingram, Jessica R.; Knockenhauer, Kevin E.; Markus, Benedikt M.; Mandelbaum, Joseph; Ramek, Alexander; Shan, Yibing; Shaw, David E.; Schwartz, Thomas U.; Ploegh, Hidde L.; Lourido, Sebastian

    2015-01-01

    Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics, the effects of 1B7–kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes. PMID:26305940

  16. Allosteric activation of apicomplexan calcium-dependent protein kinases

    SciTech Connect

    Ingram, Jessica R.; Knockenhauer, Kevin E.; Markus, Benedikt M.; Mandelbaum, Joseph; Ramek, Alexander; Shan, Yibing; Shaw, David E.; Schwartz, Thomas U.; Ploegh, Hidde L.; Lourido, Sebastian

    2015-08-24

    Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics, the effects of 1B7–kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.

  17. Allosteric activation of apicomplexan calcium-dependent protein kinases

    DOE PAGES

    Ingram, Jessica R.; Knockenhauer, Kevin E.; Markus, Benedikt M.; ...

    2015-08-24

    Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics,more » the effects of 1B7–kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.« less

  18. Homology modeling of yeast cyclin-dependent protein kinase

    NASA Astrophysics Data System (ADS)

    Selwyne, R. A.; Kholmurodov, Kh. T.; Koltovaya, N. A.

    2007-07-01

    The important functions that CDKs perform in cell division and cell cycle regulation made central protein kinase of Saccharomyces cerevisiae CDC28 a target model for structural and functional analysis. The 3D models of CDC28 protein kinase using molecular modeling techniques will enlarge our understanding of the phosphorylation mechanism and the structural changes of mutant kinases. The structural template for S. cerevisiae CDC28 was identified from PDB (Protein Databank) using BLASTP (basic local alignment search tool for proteins). Template-target alignments were generated for homology modeling and checked manually for errors. The models were then generated using MODELLER and validated using PROCHECK followed by energy minimization and molecular dynamics calculations in AMBER force field.

  19. Differential regulation of the mitogen-activated protein and stress-activated protein kinase cascades by adrenergic agonists in quiescent and regenerating adult rat hepatocytes.

    PubMed Central

    Spector, M S; Auer, K L; Jarvis, W D; Ishac, E J; Gao, B; Kunos, G; Dent, P

    1997-01-01

    To study the mechanisms by which catecholamines regulate hepatocyte proliferation after partial hepatectomy (PHX), hepatocytes were isolated from adult male rats 24 h after sham operation or two-thirds PHX and treated with catecholamines and other agonists. In freshly isolated sham cells, p42 mitogen-activated protein (MAP) kinase activity was stimulated by the alpha1-adrenergic agonist phenylephrine (PHE). Activation of p42 MAP kinase by growth factors was blunted by pretreatment of sham hepatocytes with glucagon but not by that with the beta2-adrenergic agonist isoproterenol (ISO). In PHX cells, the ability of PHE to activate p42 MAP kinase was dramatically reduced, whereas ISO became competent to inhibit p42 MAP kinase activation. PHE treatment of sham but not PHX and ISO treatment of PHX but not sham hepatocytes also activated the stress-activated protein (SAP) kinases p46/54 SAP kinase and p38 SAP kinase. These data demonstrate that an alpha1- to beta2-adrenergic receptor switch occurs upon PHX and results in an increase in SAP kinase versus MAP kinase signaling by catecholamines. In primary cultures of hepatocytes, ISO treatment of PHX but not sham cells inhibited [3H]thymidine incorporation. In contrast, PHE treatment of sham but not PHX cells stimulated [3H]thymidine incorporation, which was reduced by approximately 25 and approximately 95% with specific inhibitors of p42 MAP kinase and p38 SAP kinase function, respectively. Inhibition of the p38 SAP kinase also dramatically reduced basal [3H]thymidine incorporation. These data suggest that p38 SAP kinase plays a permissive role in liver regeneration. Alterations in the abilities of catecholamines to modulate the activities of protein kinase A and the MAP and SAP kinase pathways may represent one physiological mechanism by which these agonists can regulate hepatocyte proliferation after PHX. PMID:9199291

  20. Light-assisted small molecule screening against protein kinases

    PubMed Central

    Inglés-Prieto, Álvaro; Reichhart, Eva; Muellner, Markus K.; Nowak, Matthias; Nijman, Sebastian M.; Grusch, Michael; Janovjak, Harald

    2015-01-01

    High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that obviates the addition of chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small molecule screen against human protein kinases including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes. PMID:26457372

  1. Regulation of blood-testis barrier by actin binding proteins and protein kinases

    PubMed Central

    Li, Nan; Tang, Elizabeth I.; Cheng, C. Yan

    2016-01-01

    The blood-testis barrier (BTB) is an important ultrastructure in the testis since the onset of spermatogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood-tissue barriers, it undergoes extensive remodeling, in particular at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule (MT)-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases. PMID:26628556

  2. Regulation of blood-testis barrier by actin binding proteins and protein kinases.

    PubMed

    Li, Nan; Tang, Elizabeth I; Cheng, C Yan

    2016-03-01

    The blood-testis barrier (BTB) is an important ultrastructure in the testis, since the onset of meiosis and spermiogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood-tissue barriers, it undergoes extensive remodeling, in particular, at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin-binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases.

  3. Evaluation of the enzyme activity of protozoan protein kinases by using an in vitro kinase assay.

    PubMed

    Kato, Kentaro

    2016-10-01

    The life cycles of parasites are more complicated than those of other biological species. Protein kinases (PKs) encoded by parasites are the main triggers of life stage conversions. Phosphorylation by cellular PKs regulates important cellular processes, and the protozoan genome contains many PKs. Some PK inhibitors inhibit specific parasite life cycle event. In this report, I present a practical approach to expressing and purifying protozoan PKs by using a wheat germ cell-free protein synthesis system and I assess the phosphorylation activities of protozoan PKs by using an in vitro kinase assay.

  4. Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors

    PubMed Central

    Ojo, Kayode K; Larson, Eric T; Keyloun, Katelyn R; Castaneda, Lisa J; DeRocher, Amy E; Inampudi, Krishna K; Kim, Jessica E; Arakaki, Tracy L; Murphy, Ryan C; Zhang, Li; Napuli, Alberto J; Maly, Dustin J; Verlinde, Christophe LMJ; Buckner, Frederick S; Parsons, Marilyn; Hol, Wim GJ; Merritt, Ethan A; Van Voorhis, Wesley C

    2010-01-01

    New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds designed to be inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site at residue 128. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii cells expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to this class of selective kinase inhibitors. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable. PMID:20436472

  5. Protein Kinases of the Hippo Pathway: Regulation and Substrates

    PubMed Central

    Avruch, Joseph; Zhou, Dawang; Fitamant, Julien; Bardeesy, Nabeel; Mou, Fan; Barrufet, Laura Regué

    2012-01-01

    The “Hippo” signaling pathway has emerged as a major regulator of cell proliferation and survival in metazoans. The pathway, as delineated by genetic and biochemical studies in Drosophila, consists of a kinase cascade regulated by cell-cell contact and cell polarity that inhibits the transcriptional coactivator Yorkie and its proliferative, anti-differentiation, antiapoptotic transcriptional program. The core pathway components are the GC kinase Hippo, which phosphorylates the noncatalytic polypeptide Mats/Mob1 and, with the assistance of the scaffold protein Salvador, phosphorylates the ndr-family kinase Lats. In turn phospho-Lats, after binding to phospho-Mats, autoactivates and phosphorylates Yorkie, resulting in its nuclear exit. Hippo also uses the scaffold protein Furry and a different Mob protein to control another ndr-like kinase, the morphogenetic regulator Tricornered. Architecturally homologous kinase cascades consisting of a GC kinase, a Mob protein, a scaffolding polypeptide and an ndr-like kinase are well described in yeast; in S. cerevisiae e.g., the MEN pathway promotes mitotic exit whereas the RAM network, using a different GC kinase, Mob protein, scaffold and ndr-like kinase, regulates cell polarity and morphogenesis. In mammals, the Hippo orthologues Mst1 and Mst2 utilize the Salvador ortholog WW45/Sav1 and other scaffolds to regulate the kinases Lats1/Lats2 and ndr1/ndr2. As in Drosophila, murine Mst1/Mst2, in a redundant manner, negatively regulate the Yorkie ortholog YAP in the epithelial cells of the liver and gut; loss of both Mst1 and Mst2 results in hyperproliferation and tumorigenesis that can be largely negated by reduction or elimination of YAP. Despite this conservation, considerable diversification in pathway composition and regulation is already evident; in skin e.g., YAP phosphorylation is independent of Mst1Mst2 and Lats1Lats2. Moreover, in lymphoid cells, Mst1/Mst2, under the control of the Rap1 GTPase and independent of YAP

  6. Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

    PubMed Central

    Frost, J A; Xu, S; Hutchison, M R; Marcus, S; Cobb, M H

    1996-01-01

    The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway. PMID:8668187

  7. Pea DNA Topoisomerase I Is Phosphorylated and Stimulated by Casein Kinase 2 and Protein Kinase C

    PubMed Central

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

    2003-01-01

    DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg2+-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants. PMID:12913165

  8. Protein-protein interactions of tandem affinity purified protein kinases from rice.

    PubMed

    Rohila, Jai S; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald L; Dardick, Christopher; Canlas, Patrick; Fujii, Hiroaki; Gribskov, Michael; Kanrar, Siddhartha; Knoflicek, Lucas; Stevenson, Becky; Xie, Mingtang; Xu, Xia; Zheng, Xianwu; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E

    2009-08-19

    Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex.

  9. Protein-Protein Interactions of Tandem Affinity Purified Protein Kinases from Rice

    PubMed Central

    Rohila, Jai S.; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald L.; Dardick, Christopher; Canlas, Patrick; Fujii, Hiroaki; Gribskov, Michael; Kanrar, Siddhartha; Knoflicek, Lucas; Stevenson, Becky; Xie, Mingtang; Xu, Xia; Zheng, Xianwu; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E.

    2009-01-01

    Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex. PMID:19690613

  10. The molecular basis of targeting protein kinases in cancer therapeutics.

    PubMed

    Tsai, Chung-Jung; Nussinov, Ruth

    2013-08-01

    In this paper, we provide an overview of targeted anticancer therapies with small molecule kinase inhibitors. First, we discuss why a single constitutively active kinase emanating from a variety of aberrant genetic alterations is capable of transforming a normal cell, leading it to acquire the hallmarks of a cancer cell. To draw attention to the fact that kinase inhibition in targeted cancer therapeutics differs from conventional cytotoxic chemotherapy, we exploit a conceptual framework explaining why suppressed kinase activity will selectively kill only the so-called oncogene 'addicted' cancer cell, while sparing the healthy cell. Second, we introduce the protein kinase superfamily in light of its common active conformation with precisely positioned structural elements, and the diversified auto-inhibitory conformations among the kinase families. Understanding the detailed activation mechanism of individual kinases is essential to relate the observed oncogenic alterations to the elevated constitutively active state, to identify the mechanism of consequent drug resistance, and to guide the development of the next-generation inhibitors. To clarify the vital importance of structural guidelines in studies of oncogenesis, we explain how somatic mutations in EGFR result in kinase constitutive activation. Third, in addition to the common theme of secondary (acquired) mutations that prevent drug binding from blocking a signaling pathway which is hijacked by the aberrant activated kinase, we discuss scenarios of drug resistance and relapse by compensating lesions that bypass the inactivated pathway in a vertical or horizontal fashion. Collectively, these suggest that the future challenge of cancer therapy with small molecule kinase inhibitors will rely on the discovery of distinct combinations of optimized drugs to target individual subtypes of different cancers.

  11. Regulation of protein kinase C by the cytoskeletal protein calponin.

    PubMed

    Leinweber, B; Parissenti, A M; Gallant, C; Gangopadhyay, S S; Kirwan-Rhude, A; Leavis, P C; Morgan, K G

    2000-12-22

    Previous studies from this laboratory have shown that, upon agonist activation, calponin co-immunoprecipitates and co-localizes with protein kinase Cepsilon (PKCepsilon) in vascular smooth muscle cells. In the present study we demonstrate that calponin binds directly to the regulatory domain of PKC both in overlay assays and, under native conditions, by sedimentation with lipid vesicles. Calponin was found to bind to the C2 region of both PKCepsilon and PKCalpha with possible involvement of C1B. The C2 region of PKCepsilon binds to the calponin repeats with a requirement for the region between amino acids 160 and 182. We have also found that calponin can directly activate PKC autophosphorylation. By using anti-phosphoantibodies to residue Ser-660 of PKCbetaII, we found that calponin, in a lipid-independent manner, increased auto-phosphorylation of PKCalpha, -epsilon, and -betaII severalfold compared with control conditions. Similarly, calponin was found to increase the amount of (32)P-labeled phosphate incorporated into PKC from [gamma-(32)P]ATP. We also observed that calponin addition strongly increased the incorporation of radiolabeled phosphate into an exogenous PKC peptide substrate, suggesting an activation of enzyme activity. Thus, these results raise the possibility that calponin may function in smooth muscle to regulate PKC activity by facilitating the phosphorylation of PKC.

  12. 5'-AMP-activated protein kinase signaling in Caenorhabditis elegans.

    PubMed

    Beale, Elmus G

    2008-01-01

    5'-AMP-activated protein kinase (AMPK) has been called "the metabolic master switch" because of its central role in regulating fuel homeostasis. AMPK, a heterotrimeric serine/threonine protein kinase composed of alpha, beta, and gamma subunits, is activated by upstream kinases and by 5'-AMP in response to various nutritional and stress signals. Downstream effects include regulation of metabolism, protein synthesis, cell growth, and mediation of the actions of a number of hormones, including leptin. However, AMPK research represents a young and growing field; hence, there are many unanswered questions regarding the control and action of AMPK. This review presents evidence for the existence of AMPK signaling pathways in Caenorhabditis elegans, a genetically tractable model organism that has yet to be fully exploited to elucidate AMPK signaling mechanisms.

  13. Cyclic AMP-dependent protein kinase activity in Trypanosoma cruzi.

    PubMed Central

    Ulloa, R M; Mesri, E; Esteva, M; Torres, H N; Téllez-Iñón, M T

    1988-01-01

    A cyclic AMP-dependent protein kinase activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns, giving two peaks of kinase activity, which were eluted at 0.15 M- and 0.32 M-NaCl respectively. The second activity peak was stimulated by nanomolar concentrations of cyclic AMP. In addition, a cyclic AMP-binding protein co-eluted with the second kinase activity peak. Cyclic AMP-dependent protein kinase activity was further purified by gel filtration, affinity chromatography on histone-agarose and cyclic AMP-agarose, as well as by chromatography on CM-Sephadex. The enzyme ('holoenzyme') could be partially dissociated into two different components: 'catalytic' and 'regulatory'. The 'regulatory' component had specific binding for cyclic AMP, and it inhibited phosphotransferase activity of the homologous 'catalytic component' or of the 'catalytic subunit' from bovine heart. Cyclic AMP reversed these inhibitions. A 'holoenzyme preparation' was phosphorylated in the absence of exogenous phosphate acceptor and analysed by polyacrylamide-gel electrophoresis. A 56 kDa band was phosphorylated. The same preparation was analysed by Western blotting, by using polyclonal antibodies to the regulatory subunits of protein kinases type I or II. Both antibodies reacted with the 56 kDa band. Images Fig. 7. Fig. 8. PMID:2848508

  14. Adenosine kinase modulates root gravitropism and cap morphogenesis in Arabidopsis.

    PubMed

    Young, Li-Sen; Harrison, Benjamin R; Narayana Murthy, U M; Moffatt, Barbara A; Gilroy, Simon; Masson, Patrick H

    2006-10-01

    Adenosine kinase (ADK) is a key enzyme that regulates intra- and extracellular levels of adenosine, thereby modulating methyltransferase reactions, production of polyamines and secondary compounds, and cell signaling in animals. Unfortunately, little is known about ADK's contribution to the regulation of plant growth and development. Here, we show that ADK is a modulator of root cap morphogenesis and gravitropism. Upon gravistimulation, soluble ADK levels and activity increase in the root tip. Mutation in one of two Arabidopsis (Arabidopsis thaliana) ADK genes, ADK1, results in cap morphogenesis defects, along with alterations in root sensitivity to gravistimulation and slower kinetics of root gravitropic curvature. The kinetics defect can be partially rescued by adding spermine to the growth medium, whereas the defects in cap morphogenesis and gravitropic sensitivity cannot. The root morphogenesis and gravitropism defects of adk1-1 are accompanied by altered expression of the PIN3 auxin efflux facilitator in the cap and decreased expression of the auxin-responsive DR5-GUS reporter. Furthermore, PIN3 fails to relocalize to the bottom membrane of statocytes upon gravistimulation. Consequently, adk1-1 roots cannot develop a lateral auxin gradient across the cap, necessary for the curvature response. Interestingly, adk1-1 does not affect gravity-induced cytoplasmic alkalinization of the root statocytes, suggesting either that ADK1 functions between cytoplasmic alkalinization and PIN3 relocalization in a linear pathway or that the pH and PIN3-relocalization responses to gravistimulation belong to distinct branches of the pathway. Our data are consistent with a role for ADK and the S-adenosyl-L-methionine pathway in the control of root gravitropism and cap morphogenesis.

  15. Replicative Functions of Minute Virus of Mice NS1 Protein Are Regulated In Vitro by Phosphorylation through Protein Kinase C

    PubMed Central

    Nüesch, Jürg P. F.; Dettwiler, Sabine; Corbau, Romuald; Rommelaere, Jean

    1998-01-01

    NS1, the major nonstructural protein of the parvovirus minute virus of mice, is a multifunctional phosphoprotein which is involved in cytotoxicity, transcriptional regulation, and initiation of viral DNA replication. For coordination of these various functions during virus propagation, NS1 has been proposed to be regulated by posttranslational modifications, in particular phosphorylation. Recent in vitro studies (J. P. F. Nüesch, R. Corbau, P. Tattersall, and J. Rommelaere, J. Virol. 72:8002–8012, 1998) provided evidence that distinct NS1 activities, notably the intrinsic helicase function, are modulated by the phosphorylation state of the protein. In order to study the dependence of the initiation of viral DNA replication on NS1 phosphorylation and to identify the protein kinases involved, we established an in vitro replication system that is devoid of endogenous protein kinases and is based on plasmid substrates containing the minimal left-end origins of replication. Cellular components necessary to drive NS1-dependent rolling-circle replication (RCR) were freed from endogenous serine/threonine protein kinases by affinity chromatography, and the eukaryotic DNA polymerases were replaced by the bacteriophage T4 DNA polymerase. While native NS1 (NS1P) supported RCR under these conditions, dephosphorylated NS1 (NS1O) was impaired. Using fractionated HeLa cell extracts, we identified two essential protein components which are able to phosphorylate NS1O, are enriched in protein kinase C (PKC), and, when present together, reactivate NS1O for replication. One of these components, containing atypical PKC, was sufficient to restore NS1O helicase activity. The requirement of NS1O reactivation for characteristic PKC cofactors such as Ca2+/phosphatidylserine or phorbol esters strongly suggests the involvement of this protein kinase family in regulation of NS1 replicative functions in vitro. PMID:9811734

  16. Atypical protein kinase Clambda binds and regulates p70 S6 kinase.

    PubMed Central

    Akimoto, K; Nakaya, M; Yamanaka, T; Tanaka, J; Matsuda, S; Weng, Q P; Avruch, J; Ohno, S

    1998-01-01

    p70 S6 kinase (p70 S6K) has been implicated in the regulation of cell cycle progression. However, the mechanism of its activation is not fully understood. In the present work, evidence is provided that an atypical protein kinase C (PKC) isotype, PKClambda, is indispensable, but not sufficient, for the activation of p70 S6K. Both the regulatory and kinase domains of PKClambda associate directly with p70 S6K. Overexpression of the kinase domain without kinase activity or the regulatory domain of PKClambda results in the suppression of the serum-induced activation of p70 S6K. In addition, two types of dominant-negative mutants of PKClambda, as well as a kinase-deficient mutant of p70 S6K, suppress serum-induced DNA synthesis and E2F activation. The overexpresion of the active form of PKClambda, however, fails to activate p70 S6K. These results suggest that PKClambda is a mediator in the regulation of p70 S6K activity and plays an important role in cell cycle progression. PMID:9761742

  17. Solution structure of the cAMP-dependent protein kinase

    SciTech Connect

    Trewhella, J.; Olah, G.A.; Walsh, D.A.; Mitchell, R.D.

    1998-12-31

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project as Los Alamos National Laboratory (LANL). Protein phosphorylation is well established as one of the most important mechanisms of signal transduction and cellular regulation. Two of the key enzymes that catalyze these phosphorylation reactions are the cAMP- (PKA) and cGMP- (PKG) dependent protein kinases. PKA has served as the prototypic model of this class of enzymes that now comprises in excess of 300 phylogenetically related proteins. A large number of these protein kinases are critical for the regulation of cell function and a full analysis of their similarities and differences is essential to understand their diverse physiological roles. The cAMP-dependent protein kinase has the subunit structure R2C2, in which C and R refer to the catalytic and regulatory subunits, respectively. The cGMP-dependent protein kinase (PKG) is highly homologous to PKA but is distinguished from it by having the regulatory and catalytic domains on a contiguous polypeptide. The studies described here use small-angle scattering and Fourier Transform InfraRed (FTIR) spectroscopy to study domain movements and conformational changes in these enzymes in different functional states in order to elucidate the molecular bases for the regulation of their activities.

  18. The Roles of NDR Protein Kinases in Hippo Signalling

    PubMed Central

    Hergovich, Alexander

    2016-01-01

    The Hippo tumour suppressor pathway has emerged as a critical regulator of tissue growth through controlling cellular processes such as cell proliferation, death, differentiation and stemness. Traditionally, the core cassette of the Hippo pathway includes the MST1/2 protein kinases, the LATS1/2 protein kinases, and the MOB1 scaffold signal transducer, which together regulate the transcriptional co-activator functions of the proto-oncoproteins YAP and TAZ through LATS1/2-mediated phosphorylation of YAP/TAZ. Recent research has identified additional kinases, such as NDR1/2 (also known as STK38/STK38L) and MAP4Ks, which should be considered as novel members of the Hippo core cassette. While these efforts helped to expand our understanding of Hippo core signalling, they also began to provide insights into the complexity and redundancy of the Hippo signalling network. Here, we focus on summarising our current knowledge of the regulation and functions of mammalian NDR kinases, discussing parallels between the NDR pathways in Drosophila and mammals. Initially, we provide a general overview of the cellular functions of NDR kinases in cell cycle progression, centrosome biology, apoptosis, autophagy, DNA damage signalling, immunology and neurobiology. Finally, we put particular emphasis on discussing NDR1/2 as YAP kinases downstream of MST1/2 and MOB1 signalling in Hippo signalling. PMID:27213455

  19. Ethanol increases affinity of protein kinase C for phosphatidylserine

    SciTech Connect

    Chin, J.H.

    1986-03-01

    Protein kinase C is a calcium-dependent enzyme that requires phospholipid for its activation. It is present in relatively high concentration in the brain and may be involved in neuronal function. The present experiments test whether the membrane disorder induced by ethanol affects the activity of kinase C by changing its interaction with membrane lipid. Fractions rich in kinase C were purified from rat brain cytosol by DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Enzyme activity was assayed by measuring the phosphorylation of histone H1. As expected, phosphatidylserine activated the enzyme, and the stimulation was further increased by the addition of calcium and/or diacylglycerol. At low concentration of free calcium (0.5-1..mu..M), ethanol (800 mM0 enhanced kinase C activity if the presence of phospholipid. similar results were observed in the absence of calcium. Double reciprocal plots of the data showed that ethanol increased the affinity of the enzyme for phosphatidylserine without affecting the V/sub max. The stimulation of kinase C activity by ethanol was not observed at high calcium concentrations. These experiments suggest that ethanol may activated protein kinase C at physiological levels of calcium by facilitating its transfer into the hydrophobic membrane environment.

  20. Bacteroides fragilis Enterotoxin Upregulates Heme Oxygenase-1 in Intestinal Epithelial Cells via a Mitogen-Activated Protein Kinase- and NF-κB-Dependent Pathway, Leading to Modulation of Apoptosis

    PubMed Central

    Ko, Su Hyuk; Rho, Da Jeong; Jeon, Jong Ik; Kim, Young-Jeon; Woo, Hyun Ae; Lee, Yun Kyung

    2016-01-01

    The Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), interacts with intestinal epithelial cells and can provoke signals that induce mucosal inflammation. Although expression of heme oxygenase-1 (HO-1) is associated with regulation of inflammatory responses, little is known about HO-1 induction in ETBF infection. This study was conducted to investigate the effect of BFT on HO-1 expression in intestinal epithelial cells. Stimulation of intestinal epithelial cells with BFT resulted in upregulated expression of HO-1. BFT activated transcription factors such as NF-κB, AP-1, and Nrf2 in intestinal epithelial cells. Upregulation of HO-1 in intestinal epithelial cells was dependent on activated IκB kinase (IKK)–NF-κB signals. However, suppression of Nrf2 or AP-1 signals in intestinal epithelial cells did not result in significant attenuation of BFT-induced HO-1 expression. HO-1 induction via IKK–NF-κB in intestinal epithelial cells was regulated by p38 mitogen-activated protein kinases (MAPKs). Furthermore, suppression of HO-1 activity led to increased apoptosis in BFT-stimulated epithelial cells. These results suggest that a signaling pathway involving p38 MAPK–IKK–NF-κB in intestinal epithelial cells is required for HO-1 induction during exposure to BFT. Following this induction, increased HO-1 expression may regulate the apoptotic process in responses to BFT stimulation. PMID:27324483

  1. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    DOE PAGES

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; ...

    2016-05-16

    The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis;more » however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.« less

  2. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    PubMed Central

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; Lopez-Garrido, Javier; Muszewska, Anna; Dudkiewicz, Małgorzata; Grynberg, Marcin; Yee, Samantha; Pogliano, Kit; Tomchick, Diana R.; Pawłowski, Krzysztof; Dixon, Jack E.; Tagliabracci, Vincent S.

    2016-01-01

    The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology. PMID:27185916

  3. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    SciTech Connect

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; Lopez-Garrido, Javier; Muszewska, Anna; Dudkiewicz, Małgorzata; Grynberg, Marcin; Yee, Samantha; Pogliano, Kit; Tomchick, Diana R.; Pawłowski, Krzysztof; Dixon, Jack E.; Tagliabracci, Vincent S.

    2016-05-16

    The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.

  4. Designed Proteins To Modulate Cellular Networks

    PubMed Central

    Cortajarena, Aitziber L.; Liu, Tina Y.; Hochstrasser, Mark; Regan, Lynne

    2012-01-01

    A major challenge of protein design is to create useful new proteins that interact specifically with biological targets in living cells. Such binding modules have many potential applications, including the targeted perturbation of protein networks. As a general approach to create such modules, we designed a library with approximately 109 different binding specificities based on a small 3-tetratricopeptide repeat (TPR) motif framework. We employed a novel strategy, based on split GFP reassembly, to screen the library for modules with the desired binding specificity. Using this approach, we identified modules that bind tightly and specifically to Dss1, a small human protein that interacts with the tumor suppressor protein BRCA2. We showed that these modules also bind the yeast homologue of Dss1, Sem1. Furthermore, we demonstrated that these modules inhibit Sem1 activity in yeast. This strategy will be generally applicable to make novel genetically encoded tools for systems/synthetic biology applications. PMID:20020775

  5. Novel adenosine 3 prime ,5 prime -cyclic monophosphate dependent protein kinases in a marine diatom

    SciTech Connect

    Lin, P.P.C.; Volcani, B.E. )

    1989-08-08

    Two novel adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) dependent protein kinases have been isolated from the diatom Cylindrotheca fusiformis. The kinases, designated I and II, are eluted from DEAE-Sephacel at 0.10 and 0.15 M NaCl. They have a high affinity for cAMP and are activated by micromolar cAMP. They exhibit maximal activity at 5 mM Mg{sup 2+} and pH 8 with the preferred phosphate donor ATP and phosphate acceptor histone H1. They phosphorylate sea urchin sperm histone H1 on a single serine site in the sequence Arg-Lys-Gly-Ser({sup 32}P)-Ser-Asn-Ala-Arg and have an apparent M{sub r} of 75,000 as determined by gel filtration and sucrose density sedimentation. In the kinase I preparation a single protein band with an apparent M{sub r} of about 78,000 is photolabeled with 8-azido({sup 32}P)cAMP and is also phosphorylated with ({gamma}-{sup 32}P)ATP in a cAMP-dependent manner, after autoradiography following sodium dodecyl sulfate gel electrophoresis. The rate of phosphorylation of the 78,000-dalton band is independent of the enzyme concentration. The results indicate that (i) these diatom cAMP-dependent protein kinases are monomeric proteins, possessing both the cAMP-binding regulatory and catalytic domains on the same polypeptide chain, (ii) the enzymes do not dissociate into smaller species upon activation by binding cAMP, and (iii) self-phosphorylation of the enzymes by an intrapeptide reaction is cAMP dependent. The two diatom cAMP kinases are refractory to the heat-stable protein kinase modulator from rabbit muscle, but they respond differently to proteolytic degradation and to inhibition by arachidonic acid and several microbial alkaloids.

  6. The regulation of AMP-activated protein kinase by phosphorylation.

    PubMed Central

    Stein, S C; Woods, A; Jones, N A; Davison, M D; Carling, D

    2000-01-01

    The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (alpha) of AMPK (Thr(172)) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr(172) on AMPK activity. Mutation of Thr(172) to an aspartic acid residue (T172D) in either alpha1 or alpha2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr(172) to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr(172) accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the alpha subunit and one site on the beta subunit. Furthermore, we provide evidence that phosphorylation of Thr(172) may be involved in the sensitivity of the AMPK complex to AMP. PMID:10642499

  7. Protein kinase CK2 inhibition down modulates the NF-κB and STAT3 survival pathways, enhances the cellular proteotoxic stress and synergistically boosts the cytotoxic effect of bortezomib on multiple myeloma and mantle cell lymphoma cells.

    PubMed

    Manni, Sabrina; Brancalion, Alessandra; Mandato, Elisa; Tubi, Laura Quotti; Colpo, Anna; Pizzi, Marco; Cappellesso, Rocco; Zaffino, Fortunato; Di Maggio, Speranza Antonia; Cabrelle, Anna; Marino, Filippo; Zambello, Renato; Trentin, Livio; Adami, Fausto; Gurrieri, Carmela; Semenzato, Gianpietro; Piazza, Francesco

    2013-01-01

    CK2 is a pivotal pro-survival protein kinase in multiple myeloma that may likely impinge on bortezomib-regulated cellular pathways. In the present study, we investigated CK2 expression in multiple myeloma and mantle cell lymphoma, two bortezomib-responsive B cell tumors, as well as its involvement in bortezomib-induced cytotoxicity and signaling cascades potentially mediating bortezomib resistance. In both tumors, CK2 expression correlated with that of its activated targets NF-κB and STAT3 transcription factors. Bortezomib-induced proliferation arrest and apoptosis were significantly amplified by the simultaneous inhibition of CK2 with two inhibitors (CX-4945 and K27) in multiple myeloma and mantle cell lymphoma cell lines, in a model of multiple myeloma bone marrow microenvironment and in cells isolated from patients. CK2 inhibition empowered bortezomib-triggered mitochondrial-dependent cell death. Phosphorylation of NF-κB p65 on Ser529 (a CK2 target site) and rise of the levels of the endoplasmic reticulum stress kinase/endoribonuclease Ire1α were markedly reduced upon CK2 inhibition, as were STAT3 phospho Ser727 levels. On the contrary, CK2 inhibition increased phospho Ser51 eIF2α levels and enhanced the bortezomib-dependent accumulation of poly-ubiquitylated proteins and of the proteotoxic stress-associated chaperone Hsp70. Our data suggest that CK2 over expression in multiple myeloma and mantle cell lymphoma cells might sustain survival signaling cascades and can antagonize bortezomib-induced apoptosis at different levels. CK2 inhibitors could be useful in bortezomib-based combination therapies.

  8. Protein Kinase CK2 Inhibition Down Modulates the NF-κB and STAT3 Survival Pathways, Enhances the Cellular Proteotoxic Stress and Synergistically Boosts the Cytotoxic Effect of Bortezomib on Multiple Myeloma and Mantle Cell Lymphoma Cells

    PubMed Central

    Manni, Sabrina; Brancalion, Alessandra; Mandato, Elisa; Tubi, Laura Quotti; Colpo, Anna; Pizzi, Marco; Cappellesso, Rocco; Zaffino, Fortunato; Di Maggio, Speranza Antonia; Cabrelle, Anna; Marino, Filippo; Zambello, Renato; Trentin, Livio; Adami, Fausto; Gurrieri, Carmela; Semenzato, Gianpietro; Piazza, Francesco

    2013-01-01

    CK2 is a pivotal pro-survival protein kinase in multiple myeloma that may likely impinge on bortezomib-regulated cellular pathways. In the present study, we investigated CK2 expression in multiple myeloma and mantle cell lymphoma, two bortezomib-responsive B cell tumors, as well as its involvement in bortezomib-induced cytotoxicity and signaling cascades potentially mediating bortezomib resistance. In both tumors, CK2 expression correlated with that of its activated targets NF-κB and STAT3 transcription factors. Bortezomib-induced proliferation arrest and apoptosis were significantly amplified by the simultaneous inhibition of CK2 with two inhibitors (CX-4945 and K27) in multiple myeloma and mantle cell lymphoma cell lines, in a model of multiple myeloma bone marrow microenvironment and in cells isolated from patients. CK2 inhibition empowered bortezomib-triggered mitochondrial-dependent cell death. Phosphorylation of NF-κB p65 on Ser529 (a CK2 target site) and rise of the levels of the endoplasmic reticulum stress kinase/endoribonuclease Ire1α were markedly reduced upon CK2 inhibition, as were STAT3 phospho Ser727 levels. On the contrary, CK2 inhibition increased phospho Ser51 eIF2α levels and enhanced the bortezomib-dependent accumulation of poly-ubiquitylated proteins and of the proteotoxic stress-associated chaperone Hsp70. Our data suggest that CK2 over expression in multiple myeloma and mantle cell lymphoma cells might sustain survival signaling cascades and can antagonize bortezomib-induced apoptosis at different levels. CK2 inhibitors could be useful in bortezomib-based combination therapies. PMID:24086494

  9. Targeting protein kinase A in cancer therapy: an update

    PubMed Central

    Sapio, Luigi; Di Maiolo, Francesca; Illiano, Michela; Esposito, Antonietta; Chiosi, Emilio; Spina, Annamaria; Naviglio, Silvio

    2014-01-01

    Protein Kinase A (PKA) is a well known member of the serine-threonin protein kinase superfamily. PKA, also known as cAMP-dependent protein kinase, is a multi-unit protein kinase that mediates signal transduction of G-protein coupled receptors through its activation upon cAMP binding. The widespread expression of PKA subunit genes, and the myriad of mechanisms by which cAMP is regulated within a cell suggest that PKA signaling is one of extreme importance to cellular function. It is involved in the control of a wide variety of cellular processes from metabolism to ion channel activation, cell growth and differentiation, gene expression and apoptosis. Importantly, since it has been implicated in the initiation and progression of many tumors, PKA has been proposed as a novel biomarker for cancer detection, and as a potential molecular target for cancer therapy. Here, we highlight some features of cAMP/PKA signaling that are relevant to cancer biology and present an update on targeting PKA in cancer therapy. PMID:26417307

  10. Comparative analysis of human and bovine protein kinases reveals unique relationship and functional diversity.

    PubMed

    Kabir, Nuzhat N; Kazi, Julhash U

    2011-10-01

    Reversible protein phosphorylation by protein kinases and phosphatases is a common event in various cellular processes. The eukaryotic protein kinase superfamily, which is one of the largest superfamilies of eukaryotic proteins, plays several roles in cell signaling and diseases. We identified 482 eukaryotic protein kinases and 39 atypical protein kinases in the bovine genome, by searching publicly accessible genetic-sequence databases. Bovines have 512 putative protein kinases, each orthologous to a human kinase. Whereas orthologous kinase pairs are, on an average, 90.6% identical, orthologous kinase catalytic domain pairs are, on an average, 95.9% identical at the amino acid level. This bioinformatic study of bovine protein kinases provides a suitable framework for further characterization of their functional and structural properties.

  11. Microfluidic IEF technique for sequential phosphorylation analysis of protein kinases

    NASA Astrophysics Data System (ADS)

    Choi, Nakchul; Song, Simon; Choi, Hoseok; Lim, Bu-Taek; Kim, Young-Pil

    2015-11-01

    Sequential phosphorylation of protein kinases play the important role in signal transduction, protein regulation, and metabolism in living cells. The analysis of these phosphorylation cascades will provide new insights into their physiological functions in many biological functions. Unfortunately, the existing methods are limited to analyze the cascade activity. Therefore, we suggest a microfluidic isoelectric focusing technique (μIEF) for the analysis of the cascade activity. Using the technique, we show that the sequential phosphorylation of a peptide by two different kinases can be successfully detected on a microfluidic chip. In addition, the inhibition assay for kinase activity and the analysis on a real sample have also been conducted. The results indicate that μIEF is an excellent means for studies on phosphorylation cascade activity.

  12. Emergency Spatiotemporal Shift: The Response of Protein Kinase D to Stress Signals in the Cardiovascular System

    PubMed Central

    Wood, Brent M.; Bossuyt, Julie

    2017-01-01

    Protein Kinase D isoforms (PKD 1-3) are key mediators of neurohormonal, oxidative, and metabolic stress signals. PKDs impact a wide variety of signaling pathways and cellular functions including actin dynamics, vesicle trafficking, cell motility, survival, contractility, energy substrate utilization, and gene transcription. PKD activity is also increasingly linked to cancer, immune regulation, pain modulation, memory, angiogenesis, and cardiovascular disease. This increasing complexity and diversity of PKD function, highlights the importance of tight spatiotemporal control of the kinase via protein–protein interactions, post-translational modifications or targeting via scaffolding proteins. In this review, we focus on the spatiotemporal regulation and effects of PKD signaling in response to neurohormonal, oxidant and metabolic signals that have implications for myocardial disease. Precise targeting of these mechanisms will be crucial in the design of PKD-based therapeutic strategies. PMID:28174535

  13. Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine

    PubMed Central

    Mao, Daniel Y.L.; Neculai, Dante; Downey, Michael; Orlicky, Stephen; Haffani, Yosr Z.; Ceccarelli, Derek F.; Ho, Jenny S.L.; Szilard, Rachel K.; Zhang, Wei; Ho, Cynthia S.; Wan, Leo; Fares, Christophe; Rumpel, Sigrun; Kurinov, Igor; Arrowsmith, Cheryl H.; Durocher, Daniel; Sicheri, Frank

    2011-01-01

    SUMMARY Kae1 is a universally conserved ATPase and part of the essential gene set in bacteria. In archaea and eukaryotes, Kae1 is embedded within the protein kinase-containing KEOPS complex. Mutation of KEOPS subunits in yeast leads to striking telomere and transcription defects but the exact biochemical function of KEOPS is not known. As a first step to elucidating its function, we solved the atomic structure of archaea-derived KEOPS complexes involving Kae1, Bud32, Pcc1 and Cgi121 subunits. Our studies suggest that Kae1 is regulated at two levels by the primordial protein kinase Bud32, which is itself regulated by Cgi121. Moreover, Pcc1 appears to function as a dimerization module, perhaps suggesting that KEOPS may be a processive molecular machine. Lastly, as Bud32 lacks the conventional substrate-recognition infrastructure of eukaryotic protein kinases including an activation segment, Bud32 may provide a glimpse of the evolutionary history of the protein kinase family. PMID:18951093

  14. Phosphorylation of phosphatase inhibitor-2 (i-2) by a bovine thymus tyrosine protein kinase, p40

    SciTech Connect

    DePaoli-Roach, A.A.; Votaw, P.; Zioncheck, T.F.; Harrison, M.L.; Geahlen, R.L.

    1987-05-01

    Phosphatase inhibitor-2, a heat stable protein of Mr 22,800, is a regulatory component of the ATP-Mg-dependent phosphatase. It has been shown that in the cell tyrosine kinase activation can result in altered phosphorylation at serine and/or threonine residues, but the mechanism involved is unknown. The authors have found that i-2 is a substrate for a tyrosine specific protein kinase, p40, purified from bovine thymus. The purified enzyme is a monomer of Mr 40,000 that is autophosphorylated at tyrosine residue(s). The stoichiometry of phosphorylation of i-2 by this tyrosine protein kinase is up to 1 mol of phosphate per mol of i-2. Phosphoaminoacid analysis revealed that all the phosphate introduced was associated with tyrosine residues. Mapping of TSP-tryptic peptides by TLE and isoelectric focusing showed one major labeled fragment. Using the ATP-Mg-dependent phosphatase, a lesser extent of phosphorylation of i-2 by p40 was obtained although partial activation of the phosphatase was observed. The effect on the activity was not due to FA/GSK-3 contamination. These results could provide an important link between tyrosine protein kinase activity and modulation of phosphorylation at serine and/or threonine residues.

  15. Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine

    SciTech Connect

    Mao, D.Y.; Neculai, D.; Downey, M.; Orlicky, S.; Haffani, Y.Z.; Ceccarelli, D.F.; Ho, J.S.; Szilard, R.K.; Zhang, W.; Ho, C.S.; Wan, L.; Fares, C.; Rumpel, S.; Kurinov, I.; Arrowsmith, C.H.; Durocher, D.; Sicheri, F.

    2009-03-27

    Kae1 is a universally conserved ATPase and part of the essential gene set in bacteria. In archaea and eukaryotes, Kae1 is embedded within the protein kinase-containing KEOPS complex. Mutation of KEOPS subunits in yeast leads to striking telomere and transcription defects, but the exact biochemical function of KEOPS is not known. As a first step to elucidating its function, we solved the atomic structure of archaea-derived KEOPS complexes involving Kae1, Bud32, Pcc1, and Cgi121 subunits. Our studies suggest that Kae1 is regulated at two levels by the primordial protein kinase Bud32, which is itself regulated by Cgi121. Moreover, Pcc1 appears to function as a dimerization module, perhaps suggesting that KEOPS may be a processive molecular machine. Lastly, as Bud32 lacks the conventional substrate-recognition infrastructure of eukaryotic protein kinases including an activation segment, Bud32 may provide a glimpse of the evolutionary history of the protein kinase family.

  16. PKA modulates GSK-3β- and cdk5-catalyzed phosphorylation of tau in site- and kinase-specific manners

    PubMed Central

    Liu, Fei; Liang, Zhihou; Shi, Jianhua; Yin, Dongmei; El-Akkad, Ezzat; Grundke-Iqbal, Inge; Iqbal, Khalid; Gong, Cheng-Xin

    2007-01-01

    Phosphorylation of tau protein is regulated by several kinases, especially glycogen synthase kinase 3β (GSK-3β), cyclin-dependent protein kinase 5 (cdk5) and cAMP-dependent protein kinase (PKA). Phosphorylation of tau by PKA primes it for phosphorylation by GSK-3β, but the site-specific modulation of GSK-3β–catalyzed tau phosphorylation by the prephosphorylation has not been well investigated. Here, we found that prephosphorylation by PKA promotes GSK-3β–catalyzed tau phosphorylation at Thr181, Ser199, Ser202, Thr205, Thr217, Thr231, Ser396 and Ser422, but inhibits its phosphorylation at Thr212 and Ser404. In contrast, the prephosphorylation had no significant effect on its subsequent phosphorylation by cdk5 at Thr181, Ser199, Thr205, Thr231 and Ser422; inhibited it at Ser202, Thr212, Thr217 and Ser404; and slightly promoted it at Ser396. These studies reveal the nature of the inter-regulation of tau phosphorylation by the three major tau kinases. PMID:17078951

  17. Networks of protein kinases and phosphatases in the individual phases of contextual fear conditioning in the C57BL/6J mouse.

    PubMed

    Mucic, Goran; Sase, Sunetra; Stork, Oliver; Lubec, Gert; Li, Lin

    2015-03-01

    Although protein kinases and phosphatases have been reported to be involved in fear memory, information about these signalling molecules in the individual phases of contextual fear conditioning (cFC) is limited. C57BL/6J mice were tested in cFC, sacrificed and hippocampi were used for screening of approximately 800 protein kinases and phosphatases by protein microarrays with subsequent Western blot confirmation of threefold higher or lower hippocampal levels as compared to foot shock controls. Immunoblotting of the protein kinases and phosphatases screened out was carried out by Western blotting. A network of protein kinases and phosphatases was generated (STRING 9.1). Animals learned the task in the paradigm and protein kinase and phosphatase levels were determined in the individual phases acquisition, consolidation and retrieval and compared to foot shock controls. Protein kinases discoidin containing receptor 2 (DDR2), mitogen activated protein kinase kinase kinase 7 (TAK1), protein phosphatases dual specificity protein phosphatase (PTEN) and protein phosphatase 2a (PP2A) were modulated in the individual phases of cFC. Phosphatidyl-inositol-3,4,5-triphosphate 3-phosphatase and phosphatidylinositol-3 kinase (PI3K) that is interacting with PTEN were modulated as well. Freezing time was correlating with PP2A levels in the retrieval phase of cFC. The abovementioned protein kinases, phosphatases and inositol-signalling enzymes were not reported so far in cFC and the results are relevant for interpretation of previous and design of future studies in cFC or fear memory. Protein phosphatase PP2A was, however, the only signalling compound tested that was directly linked to retrieval in the cFC.

  18. Hydrogen sulfide inhibits high glucose-induced matrix protein synthesis by activating AMP-activated protein kinase in renal epithelial cells.

    PubMed

    Lee, Hak Joo; Mariappan, Meenalakshmi M; Feliers, Denis; Cavaglieri, Rita C; Sataranatarajan, Kavithalakshmi; Abboud, Hanna E; Choudhury, Goutam Ghosh; Kasinath, Balakuntalam S

    2012-02-10

    Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H(2)S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase β, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase β inhibitor, had the same effect. Renal cortical content of cystathionine β-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.

  19. Mitogen-Activated Protein Kinases and Mitogen Kinase Phosphatase 1: A Critical Interplay in Macrophage Biology

    PubMed Central

    Lloberas, Jorge; Valverde-Estrella, Lorena; Tur, Juan; Vico, Tania; Celada, Antonio

    2016-01-01

    Macrophages are necessary in multiple processes during the immune response or inflammation. This review emphasizes the critical role of the mitogen-activated protein kinases (MAPKs) and mitogen kinase phosphatase-1 (MKP-1) in the functional activities of macrophages. While the phosphorylation of MAPKs is required for macrophage activation or proliferation, MKP-1 dephosphorylates these kinases, thus playing a balancing role in the control of macrophage behavior. MKP-1 is a nuclear-localized dual-specificity phosphatase whose expression is regulated at multiple levels, including at the transcriptional and post-transcriptional level. The regulatory role of MKP-1 in the interplay between MAPK phosphorylation/dephosphorylation makes this molecule a critical regulator of macrophage biology and inflammation. PMID:27446931

  20. Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase

    PubMed Central

    Jiang, Youwei; Cypess, Aaron M.; Muse, Evan D.; Wu, Cui-Rong; Unson, Cecilia G.; Merrifield, R. B.; Sakmar, Thomas P.

    2001-01-01

    We prepared a stable cell line expressing the glucagon receptor to characterize the effect of Gs-coupled receptor stimulation on extracellular signal-regulated protein kinase 1/2 (ERK1/2) activity. Glucagon treatment of the cell line caused a dose-dependent increase in cAMP concentration, activation of cAMP-dependent protein kinase (PKA), and transient release of intracellular calcium. Glucagon treatment also caused rapid dose-dependent phosphorylation and activation of mitogen-activated protein kinase kinase/ERK kinase (MEK1/2) and ERK1/2. Inhibition of either PKA or MEK1/2 blocked ERK1/2 activation by glucagon. However, no significant activation of several upstream activators of MEK, including Ras, Rap1, and Raf, was observed in response to glucagon treatment. In addition, chelation of intracellular calcium reduced glucagon-mediated ERK1/2 activation. In transient transfection experiments, glucagon receptor mutants that bound glucagon but failed to increase intracellular cAMP and calcium concentrations showed no glucagon-stimulated ERK1/2 phosphorylation. We conclude that glucagon-induced MEK1/2 and ERK1/2 activation is mediated by PKA and that an increase in intracellular calcium concentration is required for maximal ERK activation. PMID:11517300

  1. Targeting Protein Kinases to Reverse Multidrug Resistance in Sarcoma

    PubMed Central

    Chen, Hua; Shen, Jacson; Choy, Edwin; Hornicek, Francis J.; Duan, Zhenfeng

    2015-01-01

    Sarcomas are a group of cancers that arise from transformed cells of mesenchymal origin. They can be classified into over 50 subtypes, accounting for approximately 1% of adult and 15% of pediatric cancers. Wide surgical resection, radiotherapy, and chemotherapy are the most common treatments for the majority of sarcomas. Among these therapies, chemotherapy can palliate symptoms and prolong life for some sarcoma patients. However, sarcoma cells can have intrinsic or acquired resistance after treatment with chemotherapeutics drugs, leading to the development of multidrug resistance (MDR). MDR attenuates the efficacy of anticancer drugs and results in treatment failure for sarcomas. Therefore, overcoming MDR is an unmet need for sarcoma therapy. Certain protein kinases demonstrate aberrant expression and/or activity in sarcoma cells, which have been found to be involved in the regulation of sarcoma cell progression, such as cell cycle, apoptosis, and survival. Inhibiting these protein kinases may not only decrease the proliferation and growth of sarcoma cells, but also reverse their resistance to chemotherapeutic drugs to subsequently reduce the doses of anticancer drugs and decrease drug side-effects. The discovery of novel strategies targeting protein kinases opens a door to a new area of sarcoma research and provides insight into the mechanisms of MDR in chemotherapy. This review will focus on the recent studies in targeting protein kinase to reverse chemotherapeutic drug resistance in sarcoma. PMID:26827688

  2. Isoform Specificity of Protein Kinase Cs in Synaptic Plasticity

    ERIC Educational Resources Information Center

    Sossin, Wayne S.

    2007-01-01

    Protein kinase Cs (PKCs) are implicated in many forms of synaptic plasticity. However, the specific isoform(s) of PKC that underlie(s) these events are often not known. We have used "Aplysia" as a model system in order to investigate the isoform specificity of PKC actions due to the presence of fewer isoforms and a large number of documented…

  3. The Two Faces of Receptor Interacting Protein Kinase-1

    PubMed Central

    Weinlich, Ricardo; Green, Douglas R.

    2014-01-01

    Receptor Interacting Protein Kinase-1 (RIPK1), a key player in inflammation and cell death, assumes opposite functions depending on the cellular context and its posttranslational modifications. Genetic evidence supported by biochemical and cellular biology approaches shed light on the circumstances in which RIPK1 promotes or inhibits these processes. PMID:25459879

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

    SciTech Connect

    Chang, Young-Chae; Cho, Hyun-Ji

    2012-06-08

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

  5. Effect of Flos carthami on stress-activated protein kinase activity in the isolated reperfused rat heart.

    PubMed

    Siow, Y L; Choy, P C; Leung, W M; O, K

    2000-04-01

    The apoptotic death of cardiomyocytes due to ischemia/reperfusion is one of the major complications of heart disease. Ischemia/reperfusion has been shown to lead to the activation of the stress-activated protein (SAP) kinases and the p38/reactivating kinase (p38/RK). In this study, the direct effect of an aqueous Flos carthami (FC) extract on SAP kinases was investigated. When isolated rat hearts were perfused by Langendorff mode with media containing FC extract prior to the induction of global ischemia and the subsequent reperfusion, SAP kinase activity was inhibited 95%. Untreated ischemic/reperfused hearts showed a 57% elevation in the activity of SAP kinase. The in vitro effect of these FC extracts on SAP kinase was also tested. At a concentration of 10 microg/ml, the aqueous FC extract resulted in 50% inhibition of SAP kinase activity in ischemic heart tissue. Our results showed that FC affected both the interaction of SAP kinase with c-jun as well as the phosphotransferase reaction. These results clearly demonstrate that extracts from Flos carthami exerted inhibitory effects on SAP kinase. The administration of the FC extract may lead to a modulation of the apoptotic effect of SAP kinase activation induced during ischemia/reperfusion.

  6. Differential modulation of Ca2+/calmodulin-dependent protein kinase II activity by regulated interactions with N-methyl-D-aspartate receptor NR2B subunits and alpha-actinin.

    PubMed

    Robison, A J; Bartlett, Ryan K; Bass, Martha A; Colbran, Roger J

    2005-11-25

    Neuronal Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) interacts with several prominent dendritic spine proteins, which have been termed CaMKII-associated proteins. The NR2B subunit of N-methyl-d-aspartate (NMDA)-type glutamate receptor, densin-180, and alpha-actinin bind comparable, approximately stoichiometric amounts of Thr(286)-autophosphorylated CaMKIIalpha, forming a ternary complex (Robison, A. J., Bass, M. A., Jiao, Y., Macmillan, L. B., Carmody, L. C., Bartlett, R. K., and Colbran, R. J. (2005) J. Biol. Chem. 280, 35329-35336), but their impacts on CaMKII function are poorly understood. Here we show that these interactions are differentially regulated and exert distinct effects on CaMKII activity. Nonphosphorylated and Thr(286)-autophosphorylated CaMKII bind to alpha-actinin with similar efficacy, but autophosphorylation at Thr(305/306) or Ca(2+)/calmodulin binding significantly reduce this binding. Moreover, alpha-actinin antagonizes CaMKII activation by Ca(2+)/calmodulin, as assessed by autophosphorylation and phosphorylation of a peptide substrate. CaMKII binding to densin (1247-1542) is partially independent of Thr(286) autophosphorylation and is unaffected by Ca(2+)-independent autophosphorylation or Ca(2+)/calmodulin. In addition, the CaMKII binding domain of densin-180 has little effect on CaMKII activity. In contrast, the interaction of CaMKIIalpha with NR2B requires either Thr(286) autophosphorylation or the binding of both Ca(2+)/calmodulin and adenine nucleotides. NR2B inhibits both the Ca(2+)/calmodulin-dependent and autonomous activities of CaMKII by a mechanism that is competitive with autocamtide-2 substrate, non-competitive with syntide-2 substrate, and uncompetitive with respect to ATP. In combination, these data suggest that dynamically regulated interactions with CaMKII-associated proteins could play pleiotropic roles in finetuning CaMKII signaling in defined subcellular compartments.

  7. AKAP (A-kinase anchoring protein) domains: beads of structure-function on the necklace of G-protein signalling.

    PubMed

    Malbon, C C; Tao, J; Shumay, E; Wang, H-Y

    2004-11-01

    AKAPs (A-kinase anchoring proteins) are members of a diverse family of scaffold proteins that minimally possess a characteristic binding domain for the RI/RII regulatory subunit of protein kinase A and play critical roles in establishing spatial constraints for multivalent signalling assemblies. Especially for G-protein-coupled receptors, the AKAPs provide an organizing centre about which various protein kinases and phosphatases can be assembled to create solid-state signalling devices that can signal, be modulated and trafficked within the cell. The structure of AKAP250 (also known as gravin or AKAP12), based on analyses of milligram quantities of recombinant protein expressed in Escherichia coli, suggests that the AKAP is probably an unordered scaffold, acting as a necklace on which 'jewels' of structure-function (e.g. the RII-binding domain) that provide docking sites on which signalling components can be assembled. Recent results suggest that AKAP250 provides not only a 'tool box' for assembling signalling elements, but may indeed provide a basis for spatial constraint observed for many signalling paradigms. The spatial dimension of the integration of cell signalling will probably reflect many functions performed by members of the AKAP family.

  8. Ten things you should know about protein kinases: IUPHAR Review 14

    PubMed Central

    Fabbro, Doriano; Cowan-Jacob, Sandra W; Moebitz, Henrik

    2015-01-01

    Many human malignancies are associated with aberrant regulation of protein or lipid kinases due to mutations, chromosomal rearrangements and/or gene amplification. Protein and lipid kinases represent an important target class for treating human disorders. This review focus on ‘the 10 things you should know about protein kinases and their inhibitors', including a short introduction on the history of protein kinases and their inhibitors and ending with a perspective on kinase drug discovery. Although the ‘10 things’ have been, to a certain extent, chosen arbitrarily, they cover in a comprehensive way the past and present efforts in kinase drug discovery and summarize the status quo of the current kinase inhibitors as well as knowledge about kinase structure and binding modes. Besides describing the potentials of protein kinase inhibitors as drugs, this review also focus on their limitations, particularly on how to circumvent emerging resistance against kinase inhibitors in oncological indications. PMID:25630872

  9. Grape seed proanthocyanidin extracts enhance endothelial nitric oxide synthase expression through 5'-AMP activated protein kinase/Surtuin 1-Krüpple like factor 2 pathway and modulate blood pressure in ouabain induced hypertensive rats.

    PubMed

    Cui, Xiaopei; Liu, Xiangju; Feng, Hua; Zhao, Shaohua; Gao, Haiqing

    2012-01-01

    Grape seed proanthocyanidin extracts (GSPE) belonging to polyphenols, possess various biological effects including anti-inflammation, anti-oxidant, anti-aging, anti-atherosclerosis, etc. GSPE is potential in regulating endothelial function. However, the underlying mechanism is not clear yet. In this study, by small interfering RNA (siRNA) knocking down, we proved that GSPE increase endothelial nitric oxide synthase (eNOS) expression in human umbilical vessel cells (HUVECs) in vitro, which was attributed to its transcription factor Krüpple like factor 2 (KLF2) induction. Furthermore, GSPE activate 5'-AMP activated protein kinase (AMPK) and increase surtuin 1 (SIRT1) protein level, critical for KLF2 induction. We also illuminated the role of GSPE in hypertension treatment. By chronic administration of GSPE in ouabain induced hypertensive rats model, we access the effect of GSPE on blood pressure regulation and the possible mechanisms involved. After 5 weeks feeding, GSPE significantly block the ouabain induced blood pressure increase. The aortic NO production impaired by ouabain was improved. In conclusion, GSPE increase eNOS expression and NO production in an AMPK/SIRT1 dependent manner through KLF2 induction, and attenuate ouabain induced hypertension.

  10. Translational Regulation of the DOUBLETIME/CKIδ/ε Kinase by LARK Contributes to Circadian Period Modulation

    PubMed Central

    Huang, Yanmei; McNeil, Gerard P.; Jackson, F. Rob

    2014-01-01

    The Drosophila homolog of Casein Kinase I δ/ε, DOUBLETIME (DBT), is required for Wnt, Hedgehog, Fat and Hippo signaling as well as circadian clock function. Extensive studies have established a critical role of DBT in circadian period determination. However, how DBT expression is regulated remains largely unexplored. In this study, we show that translation of dbt transcripts are directly regulated by a rhythmic RNA-binding protein (RBP) called LARK (known as RBM4 in mammals). LARK promotes translation of specific alternative dbt transcripts in clock cells, in particular the dbt-RC transcript. Translation of dbt-RC exhibits circadian changes under free-running conditions, indicative of clock regulation. Translation of a newly identified transcript, dbt-RE, is induced by light in a LARK-dependent manner and oscillates under light/dark conditions. Altered LARK abundance affects circadian period length, and this phenotype can be modified by different dbt alleles. Increased LARK delays nuclear degradation of the PERIOD (PER) clock protein at the beginning of subjective day, consistent with the known role of DBT in PER dynamics. Taken together, these data support the idea that LARK influences circadian period and perhaps responses of the clock to light via the regulated translation of DBT. Our study is the first to investigate translational control of the DBT kinase, revealing its regulation by LARK and a novel role of this RBP in Drosophila circadian period modulation. PMID:25211129

  11. Specific inhibition of AGC protein kinases by antibodies against C-terminal epitopes.

    PubMed

    Traincard, François; Giacomoni, Véronique; Veron, Michel

    2004-08-13

    The sequences contributing to the catalytic site of protein kinases are not all comprised within the highly conserved catalytic core. Thus, in mammalian cAMP-dependent protein kinase (PKA), the C-terminal sequence participates in substrate binding. Using synthetic peptides mimicking the FxxF motif present at most C-termini of AGC kinases, we have raised highly specific antibodies which are potent and specific inhibitors of the catalytic activity of the cognate protein kinase. Taking into account the structure of PKA, these results point to the potential of the C-terminal region of protein kinases as a target for designing specific protein kinase inhibitors.

  12. Molecular basis of cardiac potassium channel stimulation by protein kinase A.

    PubMed Central

    Huang, X Y; Morielli, A D; Peralta, E G

    1994-01-01

    Cardiac beta-adrenergic receptors accelerate heart rate by modulating ionic currents through a pathway involving cyclic AMP-dependent protein kinase A (PKA). Previous studies have focused on the regulation of Ca2+ channels by PKA; however, due to the heterogeneity of K+ channels expressed within the heart, little is known about the mechanism by which PKA modulates individual K+ channels. Here we report that PKA strongly enhanced the activity of a cloned delayed rectifier K+ channel that is normally expressed in cardiac atria. This effect required a single PKA consensus phosphorylation site located near the amino terminus of the channel protein. Furthermore, patch clamp analysis revealed that PKA phosphorylation increased the open time that single channels spend in higher conductance states. These studies provide evidence that hormonal modulation of a cardiac K+ channel involves direct phosphorylation by PKA. PMID:8290574

  13. Protein Kinase D Regulates RhoA Activity via Rhotekin Phosphorylation*

    PubMed Central

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

    2012-01-01

    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

  14. Effects of butyltins on mitogen-activated-protein kinase kinase kinase and Ras activity in human natural killer cells.

    PubMed

    Celada, Lindsay J; Whalen, Margaret M

    2014-09-01

    Butyltins (BTs) contaminate the environment and are found in human blood. BTs, tributyltin (TBT) and dibutyltin (DBT) diminish the cytotoxic function and levels of key proteins of human natural killer (NK) cells. NK cells are an initial immune defense against tumors, virally infected cells and antibody-coated cells and thus critical to human health. The signaling pathways that regulate NK cell functions include mitogen-activated protein kinases (MAPKs). Studies have shown that exposure to BTs leads to activation of specific MAPKs and MAPK kinases (MAP2Ks) in human NK cells. MAP2K kinases (MAP3Ks) are upstream activators of MAP2Ks, which then activate MAPKs. The current study examined if BT-induced activation of MAP3Ks was responsible for MAP2K and thus, MAPK activation. This study examines the effects of TBT and DBT on the total levels of two MAP3Ks, c-Raf and ASK1, as well as activating and inhibitory phosphorylation sites on these MAP3Ks. In addition, the immediate upstream activator of c-Raf, Ras, was examined for BT-induced alterations. Our results show significant activation of the MAP3K, c-Raf, in human NK cells within 10 min of TBT exposure and the MAP3K, ASK1, after 1 h exposures to TBT. In addition, our results suggest that both TBT and DBT affect the regulation of c-Raf.

  15. Mitochondrial protein import under kinase surveillance

    PubMed Central

    Opalińska, Magdalena; Meisinger, Chris

    2014-01-01

    Despite the simplicity of the yeast Saccharomyces cerevisiae, its basic cellular machinery tremendously mirrors that of higher eukaryotic counterparts. Thus, this unicellular organism turned out to be an invaluable model system to study the countless mechanisms that govern life of the cell. Recently, it has also enabled the deciphering of signalling pathways that control flux of mitochondrial proteins to the organelle according to metabolic requirements. For decades mitochondria were considered autonomous organelles that are only partially incorporated into cellular signalling networks. Consequently, only little has been known about the role of reversible phosphorylation as a meaningful mechanism that orchestrates mitochondrial biology accordingly to cellular needs. Therefore, research in this direction has been vastly neglected. However, findings over the past few years have changed this view and new exciting fields in mitochondrial biology have emerged. Here, we summarize recent discoveries in the yeast model system that point towards a vital role of reversible phosphorylation in regulation of mitochondrial protein import. PMID:28357222

  16. Isolation of Drosophila genes encoding G protein-coupled receptor kinases.

    PubMed Central

    Cassill, J A; Whitney, M; Joazeiro, C A; Becker, A; Zuker, C S

    1991-01-01

    G protein-coupled receptors are regulated via phosphorylation by a variety of protein kinases. Recently, termination of the active state of two such receptors, the beta-adrenergic receptor and rhodopsin, has been shown to be mediated by agonist- or light-dependent phosphorylation of the receptor by members of a family of protein-serine/threonine kinases (here referred to as G protein-coupled receptor kinases). We now report the isolation of a family of genes encoding a set of Drosophila protein kinases that appear to code for G protein-coupled receptor kinases. These proteins share a high degree of sequence homology with the bovine beta-adrenergic receptor kinase. The presence of a conserved family of G protein-coupled receptor kinases in vertebrates and invertebrates points to the central role of these kinases in signal transduction cascades. Images PMID:1662381

  17. Specific dephosphorylation of phosphoproteins by protein-serine and -tyrosine kinases.

    PubMed

    Kole, H K; Abdel-Ghany, M; Racker, E

    1988-08-01

    Five protein kinases are shown to serve as specific phosphatases in the absence of ADP. Although the rates of hydrolysis are very slow compared to the forward phosphorylation rates under optimal conditions, they are of the same order as the reverse reaction in the presence of ADP. Because cells contain approximately equal to 3 mM ATP, neither the reverse reaction nor the phosphatase is likely to play a physiological role. beta-casein B phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A) is specifically dephosphorylated by protein kinase A but not by polypeptide-dependent protein kinase (protein kinase P). beta-casein B phosphorylated by protein kinase P is specifically dephosphorylated by protein kinase P but not by protein kinase A. Histone H1 phosphorylated by protein kinase C is dephosphorylated by the same enzyme in the absence of ADP. In all cases tested addition of ADP and F1-ATPase accelerates moderately the rate of dephosphorylation. Native H+-ATPase from yeast plasma membranes is isolated mainly in the phosphorylated form. It is dephosphorylated and rephosphorylated by protein kinase P but not by protein kinase A. Protein-tyrosine kinase of the epidermal growth factor receptor phosphorylates the random synthetic polypeptide poly(Glu80Tyr20). The phosphorylated polymer is specifically dephosphorylated in the absence of ADP by epidermal growth factor receptor preparations but not by insulin receptor preparations. The same polymer phosphorylated by insulin receptor is dephosphorylated by insulin receptor but not by epidermal growth factor receptor preparations. By using a cycle of dephosphorylation-rephosphorylation, it is possible to identify proteins that are phosphorylated by these protein kinases in vivo. Should this method be applicable to additional protein kinases, it should be possible to estimate the quantitative contribution of each protein kinase to a single phosphoprotein.

  18. Specific dephosphorylation of phosphoproteins by protein-serine and -tyrosine kinases.

    PubMed Central

    Kole, H K; Abdel-Ghany, M; Racker, E

    1988-01-01

    Five protein kinases are shown to serve as specific phosphatases in the absence of ADP. Although the rates of hydrolysis are very slow compared to the forward phosphorylation rates under optimal conditions, they are of the same order as the reverse reaction in the presence of ADP. Because cells contain approximately equal to 3 mM ATP, neither the reverse reaction nor the phosphatase is likely to play a physiological role. beta-casein B phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A) is specifically dephosphorylated by protein kinase A but not by polypeptide-dependent protein kinase (protein kinase P). beta-casein B phosphorylated by protein kinase P is specifically dephosphorylated by protein kinase P but not by protein kinase A. Histone H1 phosphorylated by protein kinase C is dephosphorylated by the same enzyme in the absence of ADP. In all cases tested addition of ADP and F1-ATPase accelerates moderately the rate of dephosphorylation. Native H+-ATPase from yeast plasma membranes is isolated mainly in the phosphorylated form. It is dephosphorylated and rephosphorylated by protein kinase P but not by protein kinase A. Protein-tyrosine kinase of the epidermal growth factor receptor phosphorylates the random synthetic polypeptide poly(Glu80Tyr20). The phosphorylated polymer is specifically dephosphorylated in the absence of ADP by epidermal growth factor receptor preparations but not by insulin receptor preparations. The same polymer phosphorylated by insulin receptor is dephosphorylated by insulin receptor but not by epidermal growth factor receptor preparations. By using a cycle of dephosphorylation-rephosphorylation, it is possible to identify proteins that are phosphorylated by these protein kinases in vivo. Should this method be applicable to additional protein kinases, it should be possible to estimate the quantitative contribution of each protein kinase to a single phosphoprotein. Images PMID:2901092

  19. Phosphorylation of the mRNA cap binding protein and eIF-4A by different protein kinases

    SciTech Connect

    Hagedorn, C.H.

    1987-05-01

    These studies were done to determine the identity of a protein kinase that phosphorylates the mRNA cap binding protein (CBP). Two chromatographic steps (dye and ligand and ion exchange HPLC) produced a 500x purification of an enzyme activity in rabbit reticulocytes that phosphorylated CBP at serine residues. Isoelectric focusing analysis of kinase treated CBP demonstrated 5 isoelectric species of which the 2 most anodic species were phosphorylated (contained /sup 32/P). This kinase activity phosphorylated CBP when it was isolated or in the eIF-4F complex. Purified protein kinase C, cAMP or cGMP dependent protein kinase, casein kinase I or II, myosin light chain kinase or insulin receptor kinase did not significantly phosphorylate isolated CBP or CBP in the eIF-4F complex. However, cAMP and cGMP dependent protein kinases and casein kinase II phosphorylated eIF-4A but did not phosphorylate the 46 kDa component of eIF-4F. cAMP dependent protein kinase phosphorylated a approx. 220 kDa protein doublet in eIF-4F preparations. These studies indicate that CBP kinase activity probably represents a previously unidentified protein kinase. In addition, eIF-4A appears to be phosphorylated by several protein kinases whereas the 46 kDa component of the eIF-4F complex was not.

  20. [Regulation of G protein-coupled receptor kinase activity].

    PubMed

    Haga, T; Haga, K; Kameyama, K; Nakata, H

    1994-09-01

    Recent progress on the activation of G protein-coupled receptor kinases is reviewed. beta-Adrenergic receptor kinase (beta ARK) is activated by G protein beta gamma -subunits, which interact with the carboxyl terminal portion of beta ARK. Muscarinic receptor m2-subtypes are phosphorylated by beta ARK1 in the central part of the third intracellular loop (I3). Phosphorylation of I3-GST fusion protein by beta ARK1 is synergistically stimulated by the beta gamma -subunits and mastoparan or a peptide corresponding to portions adjacent to the transmembrane segments of m2-receptors or by beta gamma -subunits and the agonist-bound I3-deleted m2 variant. These results indicate that agonist-bound receptors serve as both substrates and activators of beta ARK.

  1. A Fluorescence-Based Thermal Shift Assay Identifies Inhibitors of Mitogen Activated Protein Kinase Kinase 4

    PubMed Central

    Krishna, Sankar N.; Luan, Chi-Hao; Mishra, Rama K.; Xu, Li; Scheidt, Karl A.; Anderson, Wayne F.; Bergan, Raymond C.

    2013-01-01

    Prostate cancer (PCa) is the second highest cause of cancer death in United States males. If the metastatic movement of PCa cells could be inhibited, then mortality from PCa could be greatly reduced. Mitogen-activated protein kinase kinase 4 (MAP2K4) has previously been shown to activate pro-invasion signaling pathways in human PCa. Recognizing that MAP2K4 represents a novel and validated therapeutic target, we sought to develop and characterize an efficient process for the identification of small molecules that target MAP2K4. Using a fluorescence-based thermal shift assay (FTS) assay, we first evaluated an 80 compound library of known kinase inhibitors, thereby identifying 8 hits that thermally stabilized MAP2K4 in a concentration dependent manner. We then developed an in vitro MAP2K4 kinase assay employing the biologically relevant downstream substrates, JNK1 and p38 MAPK, to evaluate kinase inhibitory function. In this manner, we validated the performance of our initial FTS screen. We next applied this approach to a 2000 compound chemically diverse library, identified 7 hits, and confirmed them in the in vitro kinase assay. Finally, by coupling our structure-activity relationship data to MAP2K4's crystal structure, we constructed a model for ligand binding. It predicts binding of our identified inhibitory compounds to the ATP binding pocket. Herein we report the creation of a robust inhibitor-screening platform with the ability to inform the discovery and design of new and potent MAP2K4 inhibitors. PMID:24339940

  2. Septin-Associated Protein Kinases in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Perez, Adam M.; Finnigan, Gregory C.; Roelants, Françoise M.; Thorner, Jeremy

    2016-01-01

    Septins are a family of eukaryotic GTP-binding proteins that associate into linear rods, which, in turn, polymerize end-on-end into filaments, and further assemble into other, more elaborate super-structures at discrete subcellular locations. Hence, septin-based ensembles are considered elements of the cytoskeleton. One function of these structures that has been well-documented in studies conducted in budding yeast Saccharomyces cerevisiae is to serve as a scaffold that recruits regulatory proteins, which dictate the spatial and temporal control of certain aspects of the cell division cycle. In particular, septin-associated protein kinases couple cell cycle progression with cellular morphogenesis. Thus, septin-containing structures serve as signaling platforms that integrate a multitude of signals and coordinate key downstream networks required for cell cycle passage. This review summarizes what we currently understand about how the action of septin-associated protein kinases and their substrates control information flow to drive the cell cycle into and out of mitosis, to regulate bud growth, and especially to direct timely and efficient execution of cytokinesis and cell abscission. Thus, septin structures represent a regulatory node at the intersection of many signaling pathways. In addition, and importantly, the activities of certain septin-associated protein kinases also regulate the state of organization of the septins themselves, creating a complex feedback loop. PMID:27847804

  3. Monoclonal antibodies against type II rat brain protein kinase

    SciTech Connect

    Nakabayashi, C.H.; Huang, K.P.

    1987-05-01

    Three monoclonal antibodies (8/1, 10/10, and 25/3) against rat brain type II protein kinase C (PKC) were used to carry out the immunochemical characterization of this kinase. These antibodies immunoprecipitated the type II PKC in a dose-dependent manner but did neither to type I nor type III isozyme. Purified type II PKC has a molecular weight of 82,000 and consists of heterogeneous isoelectric point species, all of which are cross reactive with these antibodies. Immunoblot analysis of the tryptic fragments from PKC revealed that all three antibodies recognized the 33-38-KDa fragments, the phospholipid/phorbol ester-binding domain, but not the 45-48-KDa fragments, the kinase catalytic domain. The immune complexes of the kinase and the antibodies retained the kinase activity which was dependent on Ca/sup 2 +/ and phosphatidylserine (PS) and further activated by diacylglycerol. With antibody 8/1, the apparent Km values of the kinase for Ca/sup 2 +/ and PS were not influenced. The initial rate and final extent of autophosphorylation were reduced. The concentration of PS required for half-maximal (/sup 3/H)phorbol 12,13-dibutyrate (PDBu) binding was increased and the total PDBu binding was reduced. In the presence of optimum concentrations of Ca/sup 2 +/ and PS, the Kd of PDBu was unaffected by the antibody but the total binding was reduced. These results demonstrate that the PS/PDBu-binding domain contains the major epitope for the antibodies and the antibody mainly influences the PS/PDBu binding to the kinase.

  4. Modulation of Neuronal Survival Factor MEF2 by Kinases in Parkinson’s Disease

    PubMed Central

    Yin, Yue; She, Hua; Li, Wenming; Yang, Qian; Guo, Shuzhong; Mao, Zixu

    2012-01-01

    Parkinson’s disease (PD) is the second most common neurodegenerative disorder due to selective death of neurons in the substantia nigra pars compacta. The cause of cell death remains largely unknown. Myocyte enhancer factor 2 (MEF2) is a group of transcriptional factors required to regulate neuronal development, synaptic plasticity, as well as survival. Recent studies show that MEF2 functions are regulated in multiple subcellular organelles and suggest that dysregulation of MEF2 plays essential roles in the pathogenesis of PD. Many kinases associated with transcription, translation, protein misfolding, autophagy, and cellular energy homeostasis are involved in the neurodegenerative process. Following the first demonstration that mitogen-activated protein kinase p38 (p38 MAPK) directly phosphorylates and activates MEF2 to promote neuronal survival, several other kinase regulators of MEF2s have been identified. These include protein kinase A and extracellular signal regulated kinase 5 as positive MEF2 regulators, and cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3β as negative regulators in response to diverse toxic signals relevant to PD. It is clear that MEF2 has emerged as a key point where survival and death signals converge to exert their regulatory effects, and dysregulation of MEF2 function in multiple subcellular organelles may underlie PD pathogenesis. Moreover, several other kinases such as leucine-rich repeat kinase 2 and PTEN-induced putative kinase 1 (PINK1) are of particular interest due to their potential interaction with MEF2. PMID:22661957

  5. Expression of aurora kinase A correlates with the Wnt-modulator RACGAP1 in gastric cancer.

    PubMed

    Bornschein, Jan; Nielitz, Jessica; Drozdov, Ignat; Selgrad, Michael; Wex, Thomas; Jechorek, Doerthe; Link, Alexander; Vieth, Michael; Malfertheiner, Peter

    2016-03-01

    Canonical Wnt signaling is involved in gastric carcinogenesis. The aim of this study was to identify the link between Wnt signaling and aurora kinase A (AURKA), a target for the treatment of gastrointestinal cancers. Publicly available microarray data were used to identify phenotype-specific protein-protein interaction (PPI) subnetworks. The in silico analysis revealed a gastric cancer-specific PPI subnetwork consisting of 2745 proteins and 50,935 interactions. We focused on the link of AURKA to a Wnt-specific interaction module consisting of 92 proteins. There was a direct association of AURKA with Rac GTPase-activating protein 1 (RACGAP1), as well as with CTNBB1 (β-catenin) and CDKN1A as second-order interactors. Differential expression analysis revealed a significant downregulation of both AURKA and RACGAP1 in gastric cancer compared to noncancer controls. Biopsies from a prospective cohort of 56 patients with gastric cancer (32 intestinal type, 24 diffuse type) and 20 noncancer controls were used for validation of the identified targets. The RT-PCR data confirmed a strong correlation of AURKA and RACGAP1 gene expression both in the tumor, the tumor-adjacent and the tumor-distant mucosa. RACGAP1 in the tumor was also associated with CTNBB1 expression, and inversely associated with CDKN1A gene expression. Immunohistochemistry confirmed expression of the RACGAP1 protein in gastric cancer and the tumor-adjacent mucosa. RACGAP1 expression was not associated with tumor stage, grading, Lauren type, Helicobacter pylori infection, or age. In conclusion, AURKA is directly associated with the expression of RACGAP1, a modulator of the canonical Wnt signaling pathway.

  6. The Phosphorylation of PDX-1 by Protein Kinase CK2 Is Crucial for Its Stability

    PubMed Central

    Klein, Sabrina; Meng, Rui; Montenarh, Mathias; Götz, Claudia

    2016-01-01

    The homeodomain protein PDX-1 is a critical regulator of pancreatic development and insulin production in pancreatic β-cells. We have recently shown that PDX-1 is a substrate of protein kinase CK2; a multifunctional protein kinase which is implicated in the regulation of various cellular aspects, such as differentiation, proliferation, and survival. The CK2 phosphorylation site of PDX-1 is located within the binding region of the E3 ubiquitin ligase adaptor protein PCIF1. To study the interaction between PDX-1 and PCIF1 we used immunofluorescence analysis, co-immunoprecipitation, GST-pull-down studies, and proximity ligation assay (PLA). For the analysis of the stability of PDX-1 we performed a cycloheximide chase. We used PDX-1 in its wild-type form as well as phosphomutants of the CK2 phosphorylation site. In pancreatic β-cells PDX-1 binds to PCIF1. The phosphorylation of PDX-1 by CK2 increases the ratio of PCIF1 bound to PDX-1. The stability of PDX-1 is extended in the absence of CK2 phosphorylation. Our results identified protein kinase CK2 as new important modulator of the stability of PDX-1. PMID:28036027

  7. Roles of phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase in the regulation of protein kinase C-alpha activation in interferon-gamma-stimulated macrophages.

    PubMed

    Hardy, Pierre-Olivier; Diallo, Tamsir O; Matte, Christine; Descoteaux, Albert

    2009-09-01

    Members of the protein kinase C (PKC) family are activated by interferon-gamma (IFN-gamma) and modulate IFN-gamma-induced cellular responses by regulating the activity of transcription factors. We previously reported that PKC-alpha enhances the ability of IFN regulatory factor-1 to transactivate the class II transactivator (CIITA) promoter IV in IFN-gamma-stimulated macrophages. In addition, we showed that IFN-gamma induces the nuclear translocation of PKC-alpha but the mechanisms for this remain to be elucidated. In this study, we sought to identify signalling pathways involved in IFN-gamma-induced activation of PKC-alpha and to characterize their potential roles in modulating IFN-gamma-induced responses in macrophages. IFN-gamma-mediated nuclear translocation of PKC-alpha was a Janus activated kinase 2 (JAK2)-independent process, which required phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK). However, PKC-alpha phosphorylation was independent of PI3K and p38 MAPK, indicating that IFN-gamma-induced phosphorylation and nuclear translocation of PKC-alpha are mediated by distinct mechanisms. In addition, inhibition of PI3K, but not of p38 MAPK, strongly impaired IFN-gamma-induced CIITA and MHC II gene expression. Finally, PKC-alpha associated with signal transducer and activator of transcription 1 (STAT1) and was required for the phosphorylation of STAT1 on serine 727 in IFN-gamma-stimulated macrophages. Taken together, our data indicate that PI3K and p38 MAPK modulate IFN-gamma-stimulated PKC-alpha nuclear translocation independently of JAK2 activity and that both PI3K and PKC-alpha are required for type IV CIITA and MHC II gene expression in IFN-gamma-stimulated macrophages.

  8. Insights into protein interaction networks reveal non-receptor kinases as significant druggable targets for psoriasis.

    PubMed

    Sundarrajan, Sudharsana; Lulu, Sajitha; Arumugam, Mohanapriya

    2015-07-25

    Psoriasis is a chronic disease of the skin characterized by hyper proliferation and inflammation of the epidermis and dermal components of the skin. T-cell-dependent inflammatory process in skin governs the pathogenesis of psoriasis. An in-silico search strategy was utilized to identify psoriatic therapeutic drug targets. The gene expression profiling of psoriatic skin identified a total of 427 differentially expressed genes (DEGs). Gene ontology investigation of DEGs identified genes involved in calcium binding, apoptosis, keratinisation, lipid transportation and homeostasis apart from immune mediated processes. The protein interaction networks identified proteins involved in various signaling mechanisms with high degree of interconnections. The gene modules derived from the main network were enriched with rich kinome. These sub-networks were dominated by the presence of non-receptor kinase family members which are major signal transmitters in immune response. The computational approach has aided in the identification of non-receptor kinases as potential targets for psoriasis drug development.

  9. Insights into the regulation of 5-HT2A serotonin receptors by scaffolding proteins and kinases.

    PubMed

    Allen, John A; Yadav, Prem N; Roth, Bryan L

    2008-11-01

    5-HT(2A) serotonin receptors are essential molecular targets for the actions of LSD-like hallucinogens and atypical antipsychotic drugs. 5-HT(2A) serotonin receptors also mediate a variety of physiological processes in peripheral and central nervous systems including platelet aggregation, smooth muscle contraction, and the modulation of mood and perception. Scaffolding proteins have emerged as important regulators of 5-HT(2A) receptors and our recent studies suggest multiple scaffolds exist for 5-HT(2A) receptors including PSD95, arrestin, and caveolin. In addition, a novel interaction has emerged between p90 ribosomal S6 kinase and 5-HT(2A) receptors which attenuates receptor signaling. This article reviews our recent studies and emphasizes the role of scaffolding proteins and kinases in the regulation of 5-HT(2A) trafficking, targeting and signaling.

  10. Mitogen-activated protein kinase cascades in signaling plant growth and development.

    PubMed

    Xu, Juan; Zhang, Shuqun

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are ubiquitous signaling modules in eukaryotes. Early research of plant MAPKs has been focused on their functions in immunity and stress responses. Recent studies reveal that they also play essential roles in plant growth and development downstream of receptor-like protein kinases (RLKs). With only a limited number of MAPK components, multiple functional pathways initiated from different receptors often share the same MAPK components or even a complete MAPK cascade. In this review, we discuss how MAPK cascades function as molecular switches in response to spatiotemporal-specific ligand-receptor interactions and the availability of downstream substrates. In addition, we discuss other possible mechanisms governing the functional specificity of plant MAPK cascades, a question central to our understanding of MAPK functions.

  11. Raf kinase inhibitory protein knockout mice: expression in the brain and olfaction deficit

    PubMed Central

    Theroux, Steven; Pereira, Mandy; Casten, Kimberly S.; Burwell, Rebecca D.; Yeung, Kam C.; Sedivy, John M.; Klysik, Jan

    2007-01-01

    Raf Kinase Inhibitory Protein (RKIP-1) is involved in the regulation of the MAP kinase, NF-κB, and GPCR signaling pathways. It is expressed in numerous tissues and cell types and orthologues have been documented throughout the animal and plant kingdoms. RKIP-1 has also been reported as an inhibitor of serine proteases, and a precursor of a neurostimulatory peptide. RKIP-1 has been implicated as a suppressor of metastases in several human cancers. We generated a knockout strain of mice to further assess RKIP-1’s function in mammals. RKIP-1 is expressed in many tissues with the highest protein levels detectable in testes and brain. In the brain, expression was ubiquitous in limbic formations, and homozygous mice developed olfaction deficits in the first year of life. We postulate that RKIP-1 may be a modulator of behavioral responses. PMID:17292798

  12. Role of Mitogen-Activated Protein Kinase Sty1 in Regulation of Eukaryotic Initiation Factor 2α Kinases in Response to Environmental Stress in Schizosaccharomyces pombe▿

    PubMed Central

    Berlanga, Juan José; Rivero, Damariz; Martín, Ruth; Herrero, Saturnino; Moreno, Sergio; de Haro, César

    2010-01-01

    The mitogen-activated protein kinase (MAPK) Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in Schizosaccharomyces pombe. In fission yeast, three distinct eukaryotic initiation factor 2α (eIF2α) kinases, two mammalian HRI-related protein kinases (Hri1 and Hri2) and the Gcn2 ortholog, regulate protein synthesis in response to cellular stress conditions. In this study, we demonstrate that both Hri1 and Hri2 exhibited an autokinase activity, specifically phosphorylated eIF2α, and functionally replaced the endogenous Saccharomyces cerevisiae Gcn2. We further show that Gcn2, but not Hri1 or Hri2, is activated early after exposure to hydrogen peroxide and methyl methanesulfonate (MMS). Cells lacking Gcn2 exhibit a later activation of Hri2. The activated MAPK Sty1 negatively regulates Gcn2 and Hri2 activities under oxidative stress but not in response to MMS. In contrast, Hri2 is the primary activated eIF2α kinase in response to heat shock. In this case, the activation of Sty1 appears to be transitory and does not contribute to the modulation of the eIF2α kinase stress pathway. In strains lacking Hri2, a type 2A protein phosphatase is activated soon after heat shock to reduce eIF2α phosphorylation. Finally, the MAPK Sty1, but not the eIF2α kinases, is essential for survival upon oxidative stress or heat shock, but not upon MMS treatment. These findings point to a regulatory coordination between the Sty1 MAPK and eIF2α kinase pathways for a particular range of stress responses. PMID:19880757

  13. Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.

    PubMed Central

    Cai, H; Smola, U; Wixler, V; Eisenmann-Tappe, I; Diaz-Meco, M T; Moscat, J; Rapp, U; Cooper, G M

    1997-01-01

    The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo. PMID:9001227

  14. Spatial distribution of protein kinase A activity during cell migration is mediated by A-kinase anchoring protein AKAP Lbc.

    PubMed

    Paulucci-Holthauzen, Adriana A; Vergara, Leoncio A; Bellot, Larry J; Canton, David; Scott, John D; O'Connor, Kathleen L

    2009-02-27

    Protein kinase A (PKA) has been suggested to be spatially regulated in migrating cells due to its ability to control signaling events that are critical for polarized actin cytoskeletal dynamics. Here, using the fluorescence resonance energy transfer-based A-kinase activity reporter (AKAR1), we find that PKA activity gradients form with the strongest activity at the leading edge and are restricted to the basal surface in migrating cells. The existence of these gradients was confirmed using immunocytochemistry using phospho-PKA substrate antibodies. This observation holds true for carcinoma cells migrating randomly on laminin-1 or stimulated to migrate on collagen I with lysophosphatidic acid. Phosphodiesterase inhibition allows the formation of PKA activity gradients; however, these gradients are no longer polarized. PKA activity gradients are not detected when a non-phosphorylatable mutant of AKAR1 is used, if PKA activity is inhibited with H-89 or protein kinase inhibitor, or when PKA anchoring is perturbed. We further find that a specific A-kinase anchoring protein, AKAP-Lbc, is a major contributor to the formation of these gradients. In summary, our data show that PKA activity gradients are generated at the leading edge of migrating cells and provide additional insight into the mechanisms of PKA regulation of cell motility.

  15. Spatial Distribution of Protein Kinase A Activity during Cell Migration Is Mediated by A-kinase Anchoring Protein AKAP Lbc*

    PubMed Central

    Paulucci-Holthauzen, Adriana A.; Vergara, Leoncio A.; Bellot, Larry J.; Canton, David; Scott, John D.; O'Connor, Kathleen L.

    2009-01-01

    Protein kinase A (PKA) has been suggested to be spatially regulated in migrating cells due to its ability to control signaling events that are critical for polarized actin cytoskeletal dynamics. Here, using the fluorescence resonance energy transfer-based A-kinase activity reporter (AKAR1), we find that PKA activity gradients form with the strongest activity at the leading edge and are restricted to the basal surface in migrating cells. The existence of these gradients was confirmed using immunocytochemistry using phospho-PKA substrate antibodies. This observation holds true for carcinoma cells migrating randomly on laminin-1 or stimulated to migrate on collagen I with lysophosphatidic acid. Phosphodiesterase inhibition allows the formation of PKA activity gradients; however, these gradients are no longer polarized. PKA activity gradients are not detected when a non-phosphorylatable mutant of AKAR1 is used, if PKA activity is inhibited with H-89 or protein kinase inhibitor, or when PKA anchoring is perturbed. We further find that a specific A-kinase anchoring protein, AKAP-Lbc, is a major contributor to the formation of these gradients. In summary, our data show that PKA activity gradients are generated at the leading edge of migrating cells and provide additional insight into the mechanisms of PKA regulation of cell motility. PMID:19106088

  16. DHEA improves glucose uptake via activations of protein kinase C and phosphatidylinositol 3-kinase.

    PubMed

    Ishizuka, T; Kajita, K; Miura, A; Ishizawa, M; Kanoh, Y; Itaya, S; Kimura, M; Muto, N; Mune, T; Morita, H; Yasuda, K

    1999-01-01

    We have examined the effect of adrenal androgen, dehydroepiandrosterone (DHEA), on glucose uptake, phosphatidylinositol (PI) 3-kinase, and protein kinase C (PKC) activity in rat adipocytes. DHEA (1 microM) provoked a twofold increase in 2-[3H]deoxyglucose (DG) uptake for 30 min. Pretreatment with DHEA increased insulin-induced 2-[3H]DG uptake without alterations of insulin specific binding and autophosphorylation of insulin receptor. DHEA also stimulated PI 3-kinase activity. [3H]DHEA bound to purified PKC containing PKC-alpha, -beta, and -gamma. DHEA provoked the translocation of PKC-beta and -zeta from the cytosol to the membrane in rat adipocytes. These results suggest that DHEA stimulates both PI 3-kinase and PKCs and subsequently stimulates glucose uptake. Moreover, to clarify the in vivo effect of DHEA on Goto-Kakizaki (GK) and Otsuka Long-Evans fatty (OLETF) rats, animal models of non-insulin-dependent diabetes mellitus (NIDDM) were treated with 0.4% DHEA for 2 wk. Insulin- and 12-O-tetradecanoyl phorbol-13-acetate-induced 2-[3H]DG uptakes of adipocytes were significantly increased, but there was no significant increase in the soleus muscles in DHEA-treated GK/Wistar or OLETF/Long-Evans Tokushima (LETO) rats when compared with untreated GK/Wistar or OLETF/LETO rats. These results indicate that in vivo DHEA treatment can result in increased insulin-induced glucose uptake in two different NIDDM rat models.

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

    PubMed Central

    Jaillais, Yvon

    2016-01-01

    The directional transport of auxin, known as polar auxin transport, 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, polar auxin transport 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, putting 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 (RLK) and two-component histidine kinase receptors in polar auxin transport, noticing that there are likely RLKs involved in coordinating 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

  18. Regulation of a plant SNF1-related protein kinase by glucose-6-phosphate

    SciTech Connect

    Toroser, D.; Plaut, Z.; Huber, S.C.

    2000-05-01

    One of the major protein kinases (PK{sub III}) that phosphorylates serine-158 of spinach sucrose-phosphate synthase (SPS), which is responsible for light/dark modulation of activity, is known to be a member of the SNF1-related family of protein kinases. In the present study, the authors have developed a fluorescence-based continuous assay for measurement of PK{sub III} activity. Using the continuous assay, along with the fixed-time-point {sup 32}P-incorporation assay, they demonstrate that PK{sub III} activity is inhibited by glucose-6-phosphate (Glc-6-P). Relative inhibition by Glc-6-P was increased by decreasing pH from 8.5 to 5.5 and by reducing the concentration of Mg{sup 2+} in the assay from 10 to 2 nM. Under likely physiological conditions (PH 7.0 and 2 mM Mg{sup 2+}), 10 nM Glc-6-P inhibited kinase activity approximately 70%. Inhibition by Glc-6-P could not be ascribed to contaminants in the commercial preparations. Other metabolites inhibited PK{sub III} in the following order: Glc-6-P > mannose-6-P, fructose-1,6P{sub 2} > ribose-5-P, 3-PGA, fructose-6-P. Inorganic phosphate, Glc, and AMP were not inhibitory, and free Glc did not reverse the inhibition by Glc-6-P. Because SNF1-related protein kinases are thought to function broadly in the regulation of enzyme activity and gene expression, Glc-6-P inhibition of PK{sub III} activity potentially provides a mechanism for metabolic regulation of the reactions catalyzed by these important protein kinases.

  19. Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia

    PubMed Central

    Green, Alexa S.; Maciel, Thiago T.; Hospital, Marie-Anne; Yin, Chae; Mazed, Fetta; Townsend, Elizabeth C.; Pilorge, Sylvain; Lambert, Mireille; Paubelle, Etienne; Jacquel, Arnaud; Zylbersztejn, Florence; Decroocq, Justine; Poulain, Laury; Sujobert, Pierre; Jacque, Nathalie; Adam, Kevin; So, Jason C. C.; Kosmider, Olivier; Auberger, Patrick; Hermine, Olivier; Weinstock, David M.; Lacombe, Catherine; Mayeux, Patrick; Vanasse, Gary J.; Leung, Anskar Y.; Moura, Ivan C.; Bouscary, Didier; Tamburini, Jerome

    2015-01-01

    ABSTRACT Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors provide short-term disease control, but relapse invariably occurs within months. Pim protein kinases are oncogenic FLT3-ITD targets expressed in AML cells. We show that increased Pim kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. Ectopic Pim-2 expression induces resistance to FLT3 inhibition in both FLT3-ITD–induced myeloproliferative neoplasm and AML models in mice. Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors. Finally, dual inhibition of FLT3 and Pim kinases eradicates FLT3-ITD+ cells including primary AML cells. Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy. PMID:26601252

  20. Mevalonate kinase deficiency: disclosing the role of mevalonate pathway modulation in inflammation.

    PubMed

    Marcuzzi, Annalisa; Crovella, Sergio; Monasta, Lorenzo; Vecchi Brumatti, Liza; Gattorno, Marco; Frenkel, Joost

    2012-01-01

    Inflammation is a highly regulated process involved both in the response to pathogens as well as in tissue homeostasis. In recent years, a complex network of proteins in charge of inflammation control has been revealed by the study of hereditary periodic fever syndromes. Most of these proteins belong to a few families and share the capability of sensing pathogen-associated and damageassociated molecular patterns. By interacting with each other, these proteins participate in the assembly of molecular platforms, called inflammasomes, which ultimately lead to the activation of cytokines, to the transcription of inflammatory genes or to the induction of cell apoptosis. Among hereditary periodic fever syndromes, mevalonate kinase deficiency (MKD) is the sole in which the phenotype did not directly associate with a deficiency of these proteins, but with a metabolic defect of the mevalonate pathway, highlighting the importance of this metabolic pathway in the inflammation control. Noteworthy, drugs acting on this pathway can greatly influence the inflammatory response. The modulation of inflammation by mevalonate pathway is of interest, since it may involve mechanisms not directly referable to inflammasomes. MKD provides a model to study these mechanisms and possibly to develop new classes of anti-inflammatory drugs.

  1. AMP-activated protein kinase and metabolic control

    PubMed Central

    Viollet, Benoit; Andreelli, Fabrizio

    2011-01-01

    AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, is a major regulator of cellular and whole-body energy homeostasis that coordinates metabolic pathways in order to balance nutrient supply with energy demand. It is now recognized that pharmacological activation of AMPK improves blood glucose homeostasis, lipid profile and blood pressure in insulin-resistant rodents. Indeed, AMPK activation mimics the beneficial effects of physical activity or those of calorie restriction by acting on multiple cellular targets. In addition it is now demonstrated that AMPK is one of the probable (albeit indirect) targets of major antidiabetic drugs including, the biguanides (metformin) and thiazolidinediones, as well as of insulin sensitizing adipokines (e.g., adiponectin). Taken together, such findings highlight the logic underlying the concept of targeting the AMPK pathway for the treatment of metabolic syndrome and type 2 diabetes. PMID:21484577

  2. Analysis of Phosphorylation of the Receptor-Like Protein Kinase HAESA during Arabidopsis Floral Abscission

    PubMed Central

    Taylor, Isaiah; Wang, Ying; Seitz, Kati; Baer, John; Bennewitz, Stefan; Mooney, Brian P.; Walker, John C.

    2016-01-01

    Receptor-like protein kinases (RLKs) are the largest family of plant transmembrane signaling proteins. Here we present functional analysis of HAESA, an RLK that regulates floral organ abscission in Arabidopsis. Through in vitro and in vivo analysis of HAE phosphorylation, we provide evidence that a conserved phosphorylation site on a region of the HAE protein kinase domain known as the activation segment positively regulates HAE activity. Additional analysis has identified another putative activation segment phosphorylation site common to multiple RLKs that potentially modulates HAE activity. Comparative analysis suggests that phosphorylation of this second activation segment residue is an RLK specific adaptation that may regulate protein kinase activity and substrate specificity. A growing number of RLKs have been shown to exhibit biologically relevant dual specificity toward serine/threonine and tyrosine residues, but the mechanisms underlying dual specificity of RLKs are not well understood. We show that a phospho-mimetic mutant of both HAE activation segment residues exhibits enhanced tyrosine auto-phosphorylation in vitro, indicating phosphorylation of this residue may contribute to dual specificity of HAE. These results add to an emerging framework for understanding the mechanisms and evolution of regulation of RLK activity and substrate specificity. PMID:26784444

  3. Positioning atypical protein kinase C isoforms in the UV-induced apoptotic signaling cascade.

    PubMed Central

    Berra, E; Municio, M M; Sanz, L; Frutos, S; Diaz-Meco, M T; Moscat, J

    1997-01-01

    Recent studies have documented the involvement of the atypical protein kinase C (aPKC) isoforms in important cellular functions such as cell proliferation and survival. Exposure of cells to a genotoxic stimulus that induces apoptosis, such as UV irradiation, leads to a profound inhibition of the atypical PKC activity in vivo. In this study, we addressed the relationship between this phenomenon and different proteins involved in the apoptotic response. We show that (i) the inhibition of the aPKC activity precedes UV-induced apoptosis; (ii) UV-induced aPKC inhibition and apoptosis are independent of p53; (iii) Bcl-2 proteins are potent modulators of aPKC activity; and (iv) the aPKCs are located upstream of the interleukin-converting enzyme-like protease system, which is required for the induction of apoptosis by both Par-4 (a selective aPKC inhibitor) and UV irradiation. We also demonstrate here that inhibition of aPKC activity leads to a decrease in mitogen-activated protein (MAP) kinase activity and simultaneously an increase in p38 activity. Both effects are critical for the induction of apoptosis in response to Par-4 expression and UV irradiation. Collectively, these results clarify the position of the aPKCs in the UV-induced apoptotic pathway and strongly suggest that MAP kinases play a role in this signaling cascade. PMID:9234692

  4. Phosphorylation of the TAL1 oncoprotein by the extracellular-signal-regulated protein kinase ERK1.

    PubMed Central

    Cheng, J T; Cobb, M H; Baer, R

    1993-01-01

    Alteration of the TAL1 gene is the most common genetic lesion found in T-cell acute lymphoblastic leukemia. TAL1 encodes phosphoproteins, pp42TAL1 and pp22TAL1, that represent phosphorylated versions of the full-length (residues 1 to 331) and truncated (residues 176 to 331) TAL1 gene products, respectively. Both proteins contain the basic helix-loop-helix motif, a DNA-binding and protein dimerization motif common to several known transcriptional regulatory factors. We now report that serine residue 122 (S122) is a major phosphorylation site of pp42TAL1 in leukemic cell lines and transfected COS1 cells. In vivo phosphorylation of S122 is induced by epidermal growth factor with a rapid time course that parallels activation of the ERK/MAP2 protein kinases. Moreover, S122 is readily phosphorylated in vitro by the extracellular signal-regulated protein kinase ERK1. These data suggest that TAL1 residue S122 serves as an in vivo substrate for ERK/MAP2 kinases such as ERK1. Therefore, S122 phosphorylation may provide a mechanism whereby the properties of TAL1 polypeptides can be modulated by extracellular stimuli. Images PMID:8423803

  5. KEAP1 loss modulates sensitivity to kinase targeted therapy in lung cancer

    PubMed Central

    Krall, Elsa B; Wang, Belinda; Munoz, Diana M; Ilic, Nina; Raghavan, Srivatsan; Niederst, Matthew J; Yu, Kristine; Ruddy, David A; Aguirre, Andrew J; Kim, Jong Wook; Redig, Amanda J; Gainor, Justin F; Williams, Juliet A; Asara, John M; Doench, John G; Janne, Pasi A; Shaw, Alice T; McDonald III, Robert E; Engelman, Jeffrey A; Stegmeier, Frank; Schlabach, Michael R; Hahn, William C

    2017-01-01

    Inhibitors that target the receptor tyrosine kinase (RTK)/Ras/mitogen-activated protein kinase (MAPK) pathway have led to clinical responses in lung and other cancers, but some patients fail to respond and in those that do resistance inevitably occurs (Balak et al., 2006; Kosaka et al., 2006; Rudin et al., 2013; Wagle et al., 2011). To understand intrinsic and acquired resistance to inhibition of MAPK signaling, we performed CRISPR-Cas9 gene deletion screens in the setting of BRAF, MEK, EGFR, and ALK inhibition. Loss of KEAP1, a negative regulator of NFE2L2/NRF2, modulated the response to BRAF, MEK, EGFR, and ALK inhibition in BRAF-, NRAS-, KRAS-, EGFR-, and ALK-mutant lung cancer cells. Treatment with inhibitors targeting the RTK/MAPK pathway increased reactive oxygen species (ROS) in cells with intact KEAP1, and loss of KEAP1 abrogated this increase. In addition, loss of KEAP1 altered cell metabolism to allow cells to proliferate in the absence of MAPK signaling. These observations suggest that alterations in the KEAP1/NRF2 pathway may promote survival in the presence of multiple inhibitors targeting the RTK/Ras/MAPK pathway. DOI: http://dx.doi.org/10.7554/eLife.18970.001 PMID:28145866

  6. Protein kinase d in the cardiovascular system: emerging roles in health and disease.

    PubMed

    Avkiran, Metin; Rowland, Alexandra J; Cuello, Friederike; Haworth, Robert S

    2008-02-01

    The protein kinase D (PKD) family is a recent addition to the calcium/calmodulin-dependent protein kinase group of serine/threonine kinases, within the protein kinase complement of the mammalian genome. Relative to their alphabetically superior cousins in the AGC group of kinases, namely the various isoforms of protein kinase A, protein kinase B/Akt, and protein kinase C, PKD family members have to date received limited attention from cardiovascular investigators. Nevertheless, increasing evidence now points toward important roles for PKD-mediated signaling pathways in the cardiovascular system, particularly in the regulation of myocardial contraction, hypertrophy and remodeling. This review provides a primer on PKD signaling, using information gained from studies in multiple cell types, and discusses recent data that suggest novel functions for PKD-mediated pathways in the heart and the circulation.

  7. The Protein Kinase RSK Family - Roles in Prostate Cancer

    DTIC Science & Technology

    2005-02-01

    of the prodrug, kaempferol 3-O-(2",3",4"-tri-O-acetyl-a-L-rhamnopyranoside)(3Ac- SL0101), a novel inhibitor of the Ser/Thr protein kinase, RSK...Y, Holman NJ, Hecht SM, Lannigan DA. Synthesis of the prodrug, kaempferol 3-O-(2", 3", 4"-tri-O-acetyl-a-L-rhamnopyranoside) (3Ac-SLO1 01), a novel

  8. Flunitrazepam rapidly reduces GABAA receptor subunit protein expression via a protein kinase C-dependent mechanism

    PubMed Central

    Johnston, Jonathan D; Price, Sally A; Bristow, David R

    1998-01-01

    Acute flunitrazepam (1 μM) exposure for 1 h reduced GABAA receptor α1 (22±4%, mean±s.e.mean) and β2/3 (21±4%) subunit protein levels in cultured rat cerebellar granule cells. This rapid decrease in subunit proteins was completely prevented by bisindolymaleimide 1 (1 μM), an inhibitor of protein kinase C, but not by N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89, 4.8 μM), an inhibitor of protein kinases A and G. These results suggest the existence of a benzodiazepine-induced mechanism to rapidly alter GABAA receptor protein expression, that appears to be dependent on protein kinase C activity. PMID:9723942

  9. Genome-Wide Identification, Evolution, and Co-expression Network Analysis of Mitogen-Activated Protein Kinase Kinase Kinases in Brachypodium distachyon

    PubMed Central

    Feng, Kewei; Liu, Fuyan; Zou, Jinwei; Xing, Guangwei; Deng, Pingchuan; Song, Weining; Tong, Wei; Nie, Xiaojun

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are the conserved and universal signal transduction modules in all eukaryotes, which play the vital roles in plant growth, development, and in response to multiple stresses. In this study, we used bioinformatics methods to identify 86 MAPKKK protein encoded by 73 MAPKKK genes in Brachypodium. Phylogenetic analysis of MAPKKK family from Arabidopsis, rice, and Brachypodium has classified them into three subfamilies, of which 28 belonged to MEKK, 52 to Raf, and 6 to ZIK subfamily, respectively. Conserved protein motif, exon-intron organization, and splicing intron phase in kinase domains supported the evolutionary relationships inferred from the phylogenetic analysis. And gene duplication analysis suggested the chromosomal segment duplication happened before the divergence of the rice and Brachypodium, while all of three tandem duplicated gene pairs happened after their divergence. We further demonstrated that the MAPKKKs have evolved under strong purifying selection, implying the conservation of them. The splicing transcripts expression analysis showed that the splicesome translating longest protein tended to be adopted. Furthermore, the expression analysis of BdMAPKKKs in different organs and development stages as well as heat, virus and drought stresses revealed that the MAPKKK genes were involved in various signaling pathways. And the circadian analysis suggested there were 41 MAPKKK genes in Brachypodium showing cycled expression in at least one condition, of which seven MAPKKK genes expressed in all conditions and the promoter analysis indicated these genes possessed many cis-acting regulatory elements involved in circadian and light response. Finally, the co-expression network of MAPK, MAPKK, and MAPKKK in Brachypodium was constructed using 144 microarray and RNA-seq datasets, and ten potential MAPK cascades pathway were predicted. To conclude, our study provided the important information for evolutionary and

  10. Protein kinase CK2: a newcomer in the 'druggable kinome'.

    PubMed

    Pagano, M A; Cesaro, L; Meggio, F; Pinna, L A

    2006-12-01

    The acronym CK2 (derived from the misnomer 'casein kinase' 2) denotes one of the most pleiotropic members of the eukaryotic protein kinase superfamily, characterized by an acidic consensus sequence in which a carboxylic acid (or pre-phosphorylated) side chain at position n+3 relative to the target serine/threonine residue plays a crucial role. The latest repertoire of CK2 substrates includes approx. 300 proteins, but the analysis of available phosphopeptide databases from different sources suggests that CK2 alone may be responsible for the generation of a much larger proportion (10-20%) of the eukaryotic phosphoproteome. Although for the time being CK2 is not included among protein kinases whose inhibitors are in clinical practice or in advanced clinical trials, evidence is accumulating that elevated CK2 constitutive activity co-operates to induce a number of pathological conditions, including cancer, infectious diseases, neurodegeneration and cardiovascular pathologies. The development and usage of cell-permeant, selective inhibitors discloses a scenario whereby CK2 plays a global anti-apoptotic role, which under special circumstances may lead to untimely and pathogenic cell survival.

  11. Synthetic phosphorylation of p38α recapitulates protein kinase activity.

    PubMed

    Chooi, K Phin; Galan, Sébastien R G; Raj, Ritu; McCullagh, James; Mohammed, Shabaz; Jones, Lyn H; Davis, Benjamin G

    2014-02-05

    Through a "tag-and-modify" protein chemical modification strategy, we site-selectively phosphorylated the activation loop of protein kinase p38α. Phosphorylation at natural (180) and unnatural (172) sites created two pure phospho-forms. p38α bearing only a single phosphocysteine (pCys) as a mimic of pThr at 180 was sufficient to switch the kinase to an active state, capable of processing natural protein substrate ATF2; 172 site phosphorylation did not. In this way, we chemically recapitulated triggering of a relevant segment of the MAPK-signaling pathway in vitro. This allowed detailed kinetic analysis of global and stoichiometric phosphorylation events catalyzed by p38α and revealed that site 180 is a sufficient activator alone and engenders dominant mono-phosphorylation activity. Moreover, a survey of kinase inhibition using inhibitors with different (Type I/II) modes (including therapeutically relevant) revealed unambiguously that Type II inhibitors inhibit phosphorylated p38α and allowed discovery of a predictive kinetic analysis based on cooperativity to distinguish Type I vs II.

  12. A cyclic nucleotide-independent protein kinase in Leishmania donovani.

    PubMed Central

    Das, S; Saha, A K; Mukhopadhyay, N K; Glew, R H

    1986-01-01

    Leishmania donovani promastigotes labelled for 2 h with 32Pi incorporated radioactivity into at least 21 different proteins, as determined by SDS/polyacrylamide-gel electrophoresis. Pulse-chase studies with 32Pi demonstrated that the labelled proteins were in a dynamic state: some radiolabelled proteins rapidly disappeared and others appeared after the chase. The possibility of an ectokinase on the parasite was examined; incubation of intact parasites for 10 min at 25 degrees C in an osmotically buffered medium containing [gamma-32P]ATP, but not [alpha-32P]ATP, resulted in the labelling of 10 different protozoal proteins, presumably localized to the surface of the organism's plasma membrane. Intact promastigotes also catalysed the transfer of 32P from [gamma-32P]ATP to histones. The histone-dependent kinase was solubilized by repeated freezing and thawing, and sonication, and purified 118-fold by chromatographing the high-speed (200,000 g, 1 h) supernatant fraction on QAE-Sephadex, Sephadex G-150 and hydroxyapatite columns. The kinase eluted as a single activity peak from all three columns. The partially purified histone-dependent kinase had the following properties: pH optimum, 7.0; optimum temperature, 37 degrees C; Km for mixed calf thymus histone, 0.15 mM; Km for ATP, 0.8 mM; preferred fractionated histone acceptors, H2b greater than H4 greater than H2a greater than H3 (H1 does not serve as an acceptor); optimum activity required 10-20 mM-Mg2+; inhibited 50-80% by 0.01 mM- and 1 mM-Ca2+; activity was not stimulated by calmodulin, cyclic AMP (1 mM) or cyclic GMP (1 mM) nor inhibited by a cyclic AMP-dependent protein kinase inhibitor (50 micrograms/assay); apparent Mr 75,000, as determined by Sephadex G-150 gel filtration chromatography; phosphorylated exclusively serine residues. Protein kinase activity was low in the early exponential phase of the growth curve and increased 6-fold upon entry into the stationary phase. PMID:3030283

  13. Activation of S6 kinase in human neutrophils by calcium pyrophosphate dihydrate crystals: protein kinase C-dependent and phosphatidylinositol-3-kinase-independent pathways.

    PubMed Central

    Tudan, C; Jackson, J K; Charlton, L; Pelech, S L; Sahl, B; Burt, H M

    1998-01-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown previously to be a central enzyme in crystal-induced neutrophil activation. Since activation of the 70 kDa S6 kinase (p70S6K) has been shown to be dependent on PI 3-kinase activation in mammalian cells, and since the former is a key enzyme in the transmission of signals to the cell nucleus, activation of p70(S6K) was investigated in crystal-stimulated neutrophils. Cytosolic fractions from calcium pyrophosphate dihydrate (CPPD)-crystal-activated neutrophils were separated by Mono Q chromatography and analysed for phosphotransferase activity using a range of substrates and probed by Western analysis using antibodies to p70(S6K) and mitogen-activated protein kinase (MAP kinase). CPPD crystals induced a robust, transient activation (peak activity at 2 min) of p70(S6K) that was fully inhibited by pretreatment with rapamycin. This is the first report of the activation of p70(S6K) in neutrophil signal transduction pathways induced by an agonist. This crystal-induced activation of p70(S6K) could also be inhibited by a protein kinase C (PKC) inhibitor (Compound 3), but not by the PI 3-kinase inhibitor wortmannin. CPPD crystals also activated the ERK1 and ERK2 forms of MAP kinase (wortmannin insensitive), PKC (Compound 3 sensitive) and protein kinase B (wortmannin sensitive) in neutrophils. These data suggest that activation of p70(S6K) may proceed through a PI 3-kinase- and protein kinase B-independent but PKC-dependent pathway in crystal-activated neutrophils. PMID:9531494

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

    PubMed

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

    2015-09-01

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

  15. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.

    PubMed Central

    Matsui, T; Amano, M; Yamamoto, T; Chihara, K; Nakafuku, M; Ito, M; Nakano, T; Okawa, K; Iwamatsu, A; Kaibuchi, K

    1996-01-01

    The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway. Images PMID:8641286

  16. Quercetin modulates toll-like receptor-mediated protein kinase signaling pathways in oxLDL-challenged human PBMCs and regulates TLR-activated atherosclerotic inflammation in hypercholesterolemic rats.

    PubMed

    Bhaskar, Shobha; Helen, A

    2016-12-01

    Toll-like receptors (TLRs) are pattern recognition receptors that have a unique and essential function in innate immunity. The effect of quercetin on TLR-mediated downstream signaling mechanism and its effect on TLR-mediated MAP kinase and Akt pathways were studied in oxLDL-stimulated hPBMCs using specific inhibitors. The pretreatment of hPBMCs with specific TLR inhibitor, CLI-095, decreased the NF-κB nuclear translocation and TNF-α release by oxLDL. When the cells treated with inhibitor and quercetin together, the inhibition was more effective. The specific inhibitor for p38 MAPK, SB203580, reduced the phosphorylated p38 level and decreased the NF-κB activation and TNF-α release by oxLDL-challenged hPBMCs. This inhibitor showed enhanced inhibition when treated with quercetin together. The inhibitors for ERK1/2, PD98059, and for JNK, SP606125, also showed inhibitory effect on NF-κB activation and TNF-α release by oxLDL-simulated hPBMCs. Quercetin supplementation enhanced the inhibition of nuclear translocation of NF-κB and the release of cytokines. TLR4 inhibition study confirmed the downstream signaling mechanism mediated by NF-κB which is involved in the oxLDL-induced inflammatory response, and quercetin suppresses the cytokine, TNF-α release by modulating TLR-NF-κB signaling pathway. In addition to NF-κB signaling pathway, inflammation induced by oxLDL was also related to the activation of p38MAPK, ERK1/2 and JNK, and Akt pathways, and the protective effect of quercetin may be also related to the inhibition of activation of these pathways. Quercetin significantly downregulated the elevated mRNA expression of TLRs and cytokine TNF-α in HCD-fed atherosclerotic rats in vivo. As quercetin possesses inhibition on both TLR-NF-κB signaling pathway and TLR-mediated MAPK pathway, it is evident that it can be used as a therapeutic agent to ameliorate atherosclerotic inflammation. Since quercetin is the major flavonoid and forms the backbone of many other

  17. Crosstalk and Signaling Switches in Mitogen-Activated Protein Kinase Cascades

    PubMed Central

    Fey, Dirk; Croucher, David R.; Kolch, Walter; Kholodenko, Boris N.

    2012-01-01

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

  18. Protein Kinase C: An Attractive Target for Cancer Therapy

    PubMed Central

    Marengo, Barbara; De Ciucis, Chiara; Ricciarelli, Roberta; Pronzato, Maria A.; Marinari, Umberto M.; Domenicotti, Cinzia

    2011-01-01

    Apoptosis plays an important role during all stages of carcinogenesis and the development of chemoresistance in tumor cells may be due to their selective defects in the intracellular signaling proteins, central to apoptotic pathways. Consequently, many studies have focused on rendering the chemotherapy more effective in order to prevent chemoresistance and pre-clinical and clinical data has suggested that protein kinase C (PKC) may represent an attractive target for cancer therapy. Therefore, a complete understanding of how PKC regulates apoptosis and chemoresistance may lead to obtaining a PKC-based therapy that is able to reduce drug dosages and to prevent the development of chemoresistance. PMID:24212628

  19. The DNA-dependent protein kinase: A multifunctional protein kinase with roles in DNA double strand break repair and mitosis.

    PubMed

    Jette, Nicholas; Lees-Miller, Susan P

    2015-03-01

    The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemistry, structure and function of DNA-PK, its roles in DNA double strand break repair and its newly described roles in mitosis and other cellular processes.

  20. Pivotal Role of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 in Inflammatory Pulmonary Diseases

    PubMed Central

    Qian, Feng; Deng, Jing; Wang, Gang; Ye, Richard D.; Christman, John W.

    2016-01-01

    Mitogen-activated protein kinase (MAPK)-activated protein kinase (MK2) is exclusively regulated by p38 MAPK in vivo. Upon activation of p38 MAPK, MK2 binds with p38 MAPK, leading to phosphorylation of TTP, Hsp27, Akt and Cdc25 that are involved in regulation of various essential cellular functions. In this review, we discuss current knowledge about molecular mechanisms of MK2 in regulation of TNF-α production, NADPH oxidase activation, neutrophil migration, and DNA-damage-induced cell cycle arrest which are involved in the molecular pathogenesis of acute lung injury, pulmonary fibrosis, and non-small-cell lung cancer. Collectively current and emerging new information indicate that developing MK2 inhibitors and blocking MK2-mediated signal pathways is a potential therapeutic strategy for treatment of inflammatory and fibrotic lung diseases and lung cancer. PMID:26119506

  1. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    PubMed Central

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching

    2016-01-01

    This study investigated the potential effects of dehydroeburicoic acid (TT), a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD)-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE) of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom) on membrane glucose transporter 4 (GLUT4) and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4) and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels), fenofibrate (Feno) (at 0.25 g/kg body weight), metformin (Metf) (at 0.3 g/kg body weight) or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%). TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase), an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK) phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator

  2. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice.

    PubMed

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching

    2016-06-03

    This study investigated the potential effects of dehydroeburicoic acid (TT), a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD)-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE) of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom) on membrane glucose transporter 4 (GLUT4) and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4) and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels), fenofibrate (Feno) (at 0.25 g/kg body weight), metformin (Metf) (at 0.3 g/kg body weight) or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%). TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase), an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK) phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator

  3. Inhibition of mitogen-activated protein kinase-extracellular signal-regulated kinase disrupts latent inhibition of cued fear conditioning in C57BL/6 mice.

    PubMed

    Lewis, Michael C; Davis, Jennifer A; Gould, Thomas J

    2004-12-01

    The mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) cascade has been implicated in a variety of associative conditioning tasks. However, the role of the MAPK-ERK cascades in modulating conditioning is less clear. The authors examined the effect of the potent and selective MAPK-ERK inhibitor SL327 on latent inhibition of cued fear conditioning. The results demonstrate that 50 mg/kg and 100 mg/kg SL327 disrupt latent inhibition of cued fear conditioning. These data provide evidence for an essential role of the MAPK-ERK cascade in tasks that modulate the strength of associative conditioning. The results are discussed in relation to the molecular mechanisms that support latent inhibition of cued fear conditioning.

  4. Modulation of protein synthesis by polyamines.

    PubMed

    Igarashi, Kazuei; Kashiwagi, Keiko

    2015-03-01

    Polyamines are ubiquitous small basic molecules that play important roles in cell growth and viability. Since polyamines mainly exist as a polyamine-RNA complex, we looked for proteins whose synthesis is preferentially stimulated by polyamines at the level of translation, and thus far identified 17 proteins in Escherichia coli and 6 proteins in eukaryotes. The mechanisms of polyamine stimulation of synthesis of these proteins were investigated. In addition, the role of eIF5A, containing hypusine formed from spermidine, on protein synthesis is described. These results clearly indicate that polyamines and eIF5A contribute to cell growth and viability through modulation of protein synthesis.

  5. Protein Kinase-A Inhibition Is Sufficient to Support Human Neural Stem Cells Self-Renewal.

    PubMed

    Georges, Pauline; Boissart, Claire; Poulet, Aurélie; Peschanski, Marc; Benchoua, Alexandra

    2015-12-01

    Human pluripotent stem cell-derived neural stem cells offer unprecedented opportunities for producing specific types of neurons for several biomedical applications. However, to achieve it, protocols of production and amplification of human neural stem cells need to be standardized, cost effective, and safe. This means that small molecules should progressively replace the use of media containing cocktails of protein-based growth factors. Here we have conducted a phenotypical screening to identify pathways involved in the regulation of hNSC self-renewal. We analyzed 80 small molecules acting as kinase inhibitors and identified compounds of the 5-isoquinolinesulfonamide family, described as protein kinase A (PKA) and protein kinase G inhibitors, as candidates to support hNSC self-renewal. Investigating the mode of action of these compounds, we found that modulation of PKA activity was central in controlling the choice between self-renewal or terminal neuronal differentiation of hNSC. We finally demonstrated that the pharmacological inhibition of PKA using the small molecule HA1004 was sufficient to support the full derivation, propagation, and long-term maintenance of stable hNSC in absence of any other extrinsic signals. Our results indicated that tuning of PKA activity is a core mechanism regulating hNSC self-renewal and differentiation and delineate the minimal culture media requirement to maintain undifferentiated hNSC in vitro.

  6. The New Role for an Old Kinase: Protein Kinase CK2 Regulates Metal Ion Transport

    PubMed Central

    Johnson, Adam J.; Wu, Ming J.

    2016-01-01

    The pleiotropic serine/threonine protein kinase CK2 was the first kinase discovered. It is renowned for its role in cell proliferation and anti-apoptosis. The complexity of this kinase is well reflected by the findings of past decades in terms of its heterotetrameric structure, subcellular location, constitutive activity and the extensive catalogue of substrates. With the advent of non-biased high-throughput functional genomics such as genome-wide deletion mutant screening, novel aspects of CK2 functionality have been revealed. Our recent discoveries using the model organism Saccharomyces cerevisiae and mammalian cells demonstrate that CK2 regulates metal toxicity. Extensive literature search reveals that there are few but elegant works on the role of CK2 in regulating the sodium and zinc channels. As both CK2 and metal ions are key players in cell biology and oncogenesis, understanding the details of CK2’s regulation of metal ion homeostasis has a direct bearing on cancer research. In this review, we aim to garner the recent data and gain insights into the role of CK2 in metal ion transport. PMID:28009816

  7. Calcium-Dependent Protein Kinase Genes in Corn Roots

    NASA Technical Reports Server (NTRS)

    Takezawa, D.; Patil, S.; Bhatia, A.; Poovaiah, B. W.

    1996-01-01

    Two cDNAs encoding Ca-2(+) - Dependent Protein Kinases (CDPKs), Corn Root Protein Kinase 1 and 2 (CRPK 1, CRPK 2) were isolated from the root tip library of corn (Zea mays L., cv. Merit) and their nucleotide sequences were determined. Deduced amino acid sequences of both the clones have features characteristic of plant CDPKS, including all 11 conserved serine/threonine kinase subdomains, a junction domain and a calmodulin-like domain with four Ca-2(+), -binding sites. Northern analysis revealed that CRPKI mRNA is preferentially expressed in roots, especially in the root tip; whereas, the expression of CRPK2 mRNA was very low in all the tissues tested. In situ hybridization experiments revealed that CRPKI mRNA is highly expressed in the root apex, as compared to other parts of the root. Partially purified CDPK from the root tip phosphorylates syntide-2, a common peptide substrate for plant CDPKs, and the phosphorylation was stimulated 7-fold by the addition of Ca-2(+). Our results show that two CDPK isoforms are expressed in corn roots and they may be involved in the Ca-2(+)-dependent signal transduction process.

  8. Activation of AMP-activated protein kinase revealed by hydrogen/deuterium exchange Mass Spectrometry

    PubMed Central

    Landgraf, Rachelle R.; Goswami, Devrishi; Rajamohan, Francis; Harris, Melissa S.; Calabrese, Matthew; Hoth, Lise R.; Magyar, Rachelle; Pascal, Bruce D.; Chalmers, Michael J.; Busby, Scott A.; Kurumbail, Ravi; Griffin, Patrick R.

    2013-01-01

    Summary AMP-Activated protein kinase (AMPK) monitors cellular energy, regulates genes involved in ATP synthesis and consumption, and is allosterically activated by nucleotides and synthetic ligands. Analysis of the intact enzyme by hydrogen/deuterium exchange mass spectrometry reveals conformational perturbations of AMPK in response to binding of nucleotides, cyclodextrin and a synthetic small molecule activator, A769662. Results from this analysis clearly show that binding of AMP leads to conformational changes primarily in the γ subunit of AMPK and subtle changes in the α and β subunits. In contrast, A769662 causes profound conformational changes in the glycogen binding module of the β subunit and in the kinase domain of the α subunit suggesting that the molecular binding site of latter resides between the α and β subunits. The distinct short and long-range perturbations induced upon binding of AMP and A769662 suggest fundamentally different molecular mechanisms for activation of AMPK by these two ligands. PMID:24076403

  9. SARS-CoV nucleocapsid protein interacts with cellular pyruvate kinase protein and inhibits its activity.

    PubMed

    Wei, Wei-Yen; Li, Hui-Chun; Chen, Chiung-Yao; Yang, Chee-Hing; Lee, Shen-Kao; Wang, Chia-Wen; Ma, Hsin-Chieh; Juang, Yue-Li; Lo, Shih-Yen

    2012-04-01

    The pathogenesis of SARS-CoV remains largely unknown. To study the function of the SARS-CoV nucleocapsid protein, we have conducted a yeast two-hybrid screening experiment to identify cellular proteins that may interact with the SARS-CoV nucleocapsid protein. Pyruvate kinase (liver) was found to interact with SARS-CoV nucleocapsid protein in this experiment. The binding domains of these two proteins were also determined using the yeast two-hybrid system. The physical interaction between the SARS-CoV nucleocapsid and cellular pyruvate kinase (liver) proteins was further confirmed by GST pull-down assay, co-immunoprecipitation assay and confocal microscopy. Cellular pyruvate kinase activity in hepatoma cells was repressed by SARS-CoV nucleocapsid protein in either transiently transfected or stably transfected cells. PK deficiency in red blood cells is known to result in human hereditary non-spherocytic hemolytic anemia. It is reasonable to assume that an inhibition of PKL activity due to interaction with SARS-CoV N protein is likely to cause the death of the hepatocytes, which results in the elevation of serum alanine aminotransferase and liver dysfunction noted in most SARS patients. Thus, our results suggest that SARS-CoV could reduce pyruvate kinase activity via its nucleocapsid protein, and this may in turn cause disease.

  10. Protein-tyrosine Phosphatase and Kinase Specificity in Regulation of SRC and Breast Tumor Kinase* ♦

    PubMed Central

    Fan, Gaofeng; Aleem, Saadat; Yang, Ming; Miller, W. Todd; Tonks, Nicholas K.

    2015-01-01

    Despite significant evidence to the contrary, the view that phosphatases are “nonspecific” still pervades the field. Systems biology approaches to defining how signal transduction pathways are integrated at the level of whole organisms also often downplay the contribution of phosphatases, defining them as “erasers” that serve merely to restore the system to its basal state. Here, we present a study that counteracts the idea of “nonspecific phosphatases.” We have characterized two structurally similar and functionally related kinases, BRK and SRC, which are regulated by combinations of activating autophosphorylation and inhibitory C-terminal sites of tyrosine phosphorylation. We demonstrated specificity at the level of the kinases in that SRMS phosphorylated the C terminus of BRK, but not SRC; in contrast, CSK is the kinase responsible for C-terminal phosphorylation of SRC, but not BRK. For the phosphatases, we observed that RNAi-mediated suppression of PTP1B resulted in opposing effects on the activity of BRK and SRC and have defined the mechanisms underlying this specificity. PTP1B inhibited BRK by directly dephosphorylating the Tyr-342 autophosphorylation site. In contrast, PTP1B potentiated SRC activity, but not by dephosphorylating SRC itself directly; instead, PTP1B regulated the interaction between CBP/PAG and CSK. SRC associated with, and phosphorylated, the transmembrane protein CBP/PAG at Tyr-317, resulting in CSK recruitment. We identified PAG as a substrate of PTP1B, and dephosphorylation abolished recruitment of the inhibitory kinase CSK. Overall, these findings illustrate how the combinatorial effects of PTKs and PTPs may be integrated to regulate signaling, with both classes of enzymes displaying exquisite specificity. PMID:25897081

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

    PubMed Central

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

    2016-01-01

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

  12. Sensitization of TRPA1 by Protein Kinase A

    PubMed Central

    Meents, Jannis E.; Fischer, Michael J. M.; McNaughton, Peter A.

    2017-01-01

    The TRPA1 ion channel is expressed in nociceptive (pain-sensitive) somatosensory neurons and is activated by a wide variety of chemical irritants, such as acrolein in smoke or isothiocyanates in mustard. Here, we investigate the enhancement of TRPA1 function caused by inflammatory mediators, which is thought to be important in lung conditions such as asthma and COPD. Protein kinase A is an important kinase acting downstream of inflammatory mediators to cause sensitization of TRPA1. By using site-directed mutagenesis, patch-clamp electrophysiology and calcium imaging we identify four amino acid residues, S86, S317, S428, and S972, as the principal targets of PKA-mediated phosphorylation and sensitization of TRPA1. PMID:28076424

  13. Focal Adhesion Kinase Modulates Cell Adhesion Strengthening via Integrin Activation

    PubMed Central

    Michael, Kristin E.; Dumbauld, David W.; Burns, Kellie L.; Hanks, Steven K.

    2009-01-01

    Focal adhesion kinase (FAK) is an essential nonreceptor tyrosine kinase regulating cell migration, adhesive signaling, and mechanosensing. Using FAK-null cells expressing FAK under an inducible promoter, we demonstrate that FAK regulates the time-dependent generation of adhesive forces. During the early stages of adhesion, FAK expression in FAK-null cells enhances integrin activation to promote integrin binding and, hence, the adhesion strengthening rate. Importantly, FAK expression regulated integrin activation, and talin was required for the FAK-dependent effects. A role for FAK in integrin activation was confirmed in human fibroblasts with knocked-down FAK expression. The FAK autophosphorylation Y397 site was required for the enhancements in adhesion strengthening and integrin-binding responses. This work demonstrates a novel role for FAK in integrin activation and the time-dependent generation of cell–ECM forces. PMID:19297531

  14. Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2

    PubMed Central

    Aragay, A. M.; Mellado, M.; Frade, J. M. R.; Martin, A. M.; Jimenez-Sainz, M. C.; Martinez-A, C.; Mayor, F.

    1998-01-01

    Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium flux responses. This desensitization correlates with the Ser/Thr phosphorylation of the receptor and with the transient translocation of the G protein-coupled receptor kinase 2 (GRK2, also called β-adrenergic kinase 1 or βARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein β-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium flux responses to MCP-1 in HEK293 cells expressing the CCR2B receptor were also markedly reduced upon cotransfection with GRK2 or the homologous kinase GRK3. Nevertheless, expression of the GRK2 dominant-negative mutant βARK-K220R did not affect the initial calcium response, but favored receptor response to a subsequent challenge by agonists. The modulation of the CCR2B receptor by GRK2 suggests an important role for this kinase in the regulation of monocyte and lymphocyte response to chemokines. PMID:9501202

  15. Identifying protein kinase-specific effectors of the osmostress response in yeast.

    PubMed

    Romanov, Natalie; Hollenstein, David Maria; Janschitz, Marion; Ammerer, Gustav; Anrather, Dorothea; Reiter, Wolfgang

    2017-03-07

    The budding yeast Saccharomyces cerevisiae reacts to increased external osmolarity by modifying many cellular processes. Adaptive signaling relies primarily on the high-osmolarity glycerol (HOG) pathway, which is closely related to the mammalian p38 mitogen-activated protein kinase (MAPK) pathway in core architecture. To identify target proteins of the MAPK Hog1, we designed a mass spectrometry-based high-throughput experiment to measure the impact of Hog1 activation or inhibition on the Scerevisiae phosphoproteome. In addition, we analyzed how deletion of RCK2, which encodes a known effector protein kinase target of Hog1, modulated osmotic stress-induced phosphorylation. Our results not only provide an overview of the diversity of cellular functions that are directly and indirectly affected by the activity of the HOG pathway but also enabled an assessment of the Hog1-independent events that occur under osmotic stress conditions. We extended the number of putative Hog1 direct targets by analyzing the modulation of motifs consisting of serine or threonine followed by a proline (S/T-P motif) and subsequently validated these with an in vivo interaction assay. Rck2 appears to act as a central hub for many Hog1-mediated secondary phosphorylation events. This study clarifies many of the direct and indirect effects of HOG signaling and its stress-adaptive functions.

  16. Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27.

    PubMed

    Freshney, N W; Rawlinson, L; Guesdon, F; Jones, E; Cowley, S; Hsuan, J; Saklatvala, J

    1994-09-23

    An IL-1-stimulated protein kinase cascade resulting in phosphorylation of the small heat shock protein hsp27 has been identified in KB cells. It is distinct from the p42 MAP kinase cascade. An upstream activator kinase phosphorylated a 40 kDa kinase (p40) upon threonine and tyrosine residues, which in turn phosphorylated a 50 kDa kinase (p50) upon threonine (and some serine) residues. p50 phosphorylated hsp27 upon serine. p40 and p50 were purified to near homogeneity. All three components were inactivated by protein phosphatase 2A, and p40 was inactivated by protein tyrosine phosphatase 1B. The substrate specificity of p40 differed from that of p42 and p54 MAP kinases. The upstream activator was not a MAP kinase kinase. p50 resembled MAPKAPK-2 and may be identical.

  17. Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells.

    PubMed Central

    Prasad, K V; Janssen, O; Kapeller, R; Raab, M; Cantley, L C; Rudd, C E

    1993-01-01

    The Src-related tyrosine kinase p59fyn(T) plays an important role in the generation of intracellular signals from the T-cell antigen receptor TCR zeta/CD3 complex. A key question concerns the nature and the binding sites of downstream components that interact with this Src-related kinase. p59fyn(T) contains Src-homology 2 and 3 domains (SH2 and SH3) with a capacity to bind to intracellular proteins. One potential downstream target is phosphatidylinositol 3-kinase (PI 3-kinase). In this study, we demonstrate that anti-CD3 and anti-Fyn immunoprecipitates possess PI 3-kinase activity as assessed by TLC and HPLC. Both free and receptor-bound p59fyn(T) were found to bind to the lipid kinase. Further, our results indicate that Src-related kinases have developed a novel mechanism to interact with PI 3-kinase. Precipitation using GST fusion proteins containing Fyn SH2, SH3, and SH2/SH3 domains revealed that PI 3-kinase bound principally to the SH3 domain of Fyn. Fyn SH3 bound directly to the p85 subunit of PI 3-kinase as expressed in a baculoviral system. Anti-CD3 crosslinking induced an increase in the detection of Fyn SH3-associated PI 3-kinase activity. Thus PI 3-kinase is a target of SH3 domains and is likely to play a major role in the signals derived from the TCR zeta/CD3-p59fyn complex. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8394019

  18. Protein kinase C-zeta and protein kinase B regulate distinct steps of insulin endocytosis and intracellular sorting.

    PubMed

    Fiory, Francesca; Oriente, Francesco; Miele, Claudia; Romano, Chiara; Trencia, Alessandra; Alberobello, Anna Teresa; Esposito, Iolanda; Valentino, Rossella; Beguinot, Francesco; Formisano, Pietro

    2004-03-19

    We have investigated the molecular mechanisms regulating insulin internalization and intracellular sorting. Insulin internalization was decreased by 50% upon incubation of the cells with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. PI3K inhibition also reduced insulin degradation and intact insulin release by 50 and 75%, respectively. Insulin internalization was reduced by antisense inhibition of protein kinase C-zeta (PKCzeta) expression and by overexpression of a dominant negative PKCzeta mutant (DN-PKCzeta). Conversely, overexpression of PKCzeta increased insulin internalization as a function of the PKCzeta levels achieved in the cells. Expression of wild-type protein kinase B (PKB)-alpha or of a constitutively active form (myr-PKB) did not significantly alter insulin internalization and degradation but produced a 100% increase of intact insulin release. Inhibition of PKB by a dominant negative mutant (DN-PKB) or by the pharmacological inhibitor ML-9 reduced intact insulin release by 75% with no effect on internalization and degradation. In addition, overexpression of Rab5 completely rescued the effect of PKCzeta inhibition on insulin internalization but not that of PKB inhibition on intact insulin recycling. Indeed, PKCzeta bound to and activated Rab5. Thus, PI3K controls different steps within the insulin endocytic itinerary. PKCzeta appears to mediate the PI3K effect on insulin internalization in a Rab5-dependent manner, whereas PKB directs intracellular sorting toward intact insulin release.

  19. Characterization of serine 916 as an in vivo autophosphorylation site for protein kinase D/Protein kinase Cmu.

    PubMed

    Matthews, S A; Rozengurt, E; Cantrell, D

    1999-09-10

    Activation of the serine kinase protein kinase D (PKD)/PKCmicro is controlled by the phosphorylation of two serine residues within its activation loop via a PKC-dependent signaling cascade. In this study we have identified the C-terminal serine 916 residue as an in vivo phosphorylation site within active PKD/PKCmu. An antibody that recognized PKD/PKCmu proteins specifically phosphorylated on the serine 916 residue was generated and used to show that phosphorylation of Ser-916 is induced by phorbol ester treatment of cells. Thus, the pS916 antibody is a useful tool to study the regulation of PKD/PKCmu activity in vivo. Antigen receptor ligation of T and B lymphocytes also induced phosphorylation of the serine 916 residue of PKD/PKCmu. Furthermore the regulatory FcgammaRIIB receptor, which mediates vital negative feedback signals to the B cell antigen receptor complex, inhibited the antigen receptor-induced activation and serine 916 phosphorylation of PKD/PKCmu. The degree of serine 916 phosphorylation during lymphocyte activation and inhibition exactly correlated with the activation status of PKD/PKCmu. Moreover, using different mutants of PKD/PKCmu, we show that serine 916 is not trans-phosphorylated by an upstream