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Sample records for rets signaling kinase

  1. Crystal Structure and Oligomeric State of the RetS Signaling Kinase Sensory Domain

    SciTech Connect

    Jing, X.; Jaw, J; Robinson, H; Schubot, F

    2010-01-01

    The opportunistic pathogen Pseudomonas aeruginosa may cause both acute and chronic-persistent infections in predisposed individuals. Acute infections require the presence of a functional type III secretion system (T3SS), whereas chronic P. aeruginosa infections are characterized by the formation of drug-resistant biofilms. The T3SS and biofilm formation are reciprocally regulated by the signaling kinases LadS, RetS, and GacS. RetS downregulates biofilm formation and upregulates expression of the T3SS through a unique mechanism. RetS forms a heterodimeric complex with GacS and thus prevents GacS autophosphorylation and downstream signaling. The signals that regulate RetS are not known but RetS possesses a distinctive periplasmic sensor domain that is believed to serve as receptor for the regulatory ligand. We have determined the crystal structure of the RetS sensory domain at 2.0 {angstrom} resolution. The structure closely resembles those of carbohydrate binding modules of other proteins, suggesting that the elusive ligands are likely carbohydrate moieties. In addition to the conserved beta-sandwich structure, the sensory domain features two alpha helices which create a unique surface topology. Protein-protein crosslinking and fluorescence energy transfer experiments also revealed that the sensory domain dimerizes with a dissociation constant of K{sub d} = 580 {+-} 50 nM, a result with interesting implications for our understanding of the underlying signaling mechanism.

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

    PubMed Central

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

    2015-01-01

    REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase required for normal development and maintenance of neurons of the central and peripheral nervous systems. Deregulation of RET and hyperactivity of the RET kinase is intimately connected to several types of human cancers, most notably thyroid cancers, making it an attractive therapeutic target for small-molecule kinase inhibitors. Novel approaches, allowing external control of the activity of RET, would be key additions to the signal transduction toolbox. In this work, photoswitchable RET kinase inhibitors based on azo-functionalized pyrazolopyrimidines were developed, enabling photonic control of RET activity. The most promising compound displays excellent switching properties and stability with good inhibitory effect towards RET in cell-free as well as live-cell assays and a significant difference in inhibitory activity between its two photoisomeric forms. As the first reported photoswitchable small-molecule kinase inhibitor, we consider the herein presented effector to be a significant step forward in the development of tools for kinase signal transduction studies with spatiotemporal control over inhibitor concentration in situ. PMID:25944708

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  4. Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase

    PubMed Central

    Moccia, Marialuisa; Liu, Qingsong; Guida, Teresa; Federico, Giorgia; Brescia, Annalisa; Zhao, Zheng; Choi, Hwan Geun; Deng, Xianming; Tan, Li; Wang, Jinhua; Billaud, Marc; Gray, Nathanael S.

    2015-01-01

    Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the ‘DFG-out’ inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the ‘gatekeeper’ V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET. PMID:26046350

  5. Exon Skipping in the RET Gene Encodes Novel Isoforms That Differentially Regulate RET Protein Signal Transduction.

    PubMed

    Gabreski, Nicole A; Vaghasia, Janki K; Novakova, Silvia S; McDonald, Neil Q; Pierchala, Brian A

    2016-07-29

    Rearranged during transfection (RET), a receptor tyrosine kinase that is activated by the glial cell line-derived neurotrophic factor family ligands (GFLs), plays a crucial role in the development and function of the nervous system and additionally is required for kidney development and spermatogenesis. RET encodes a transmembrane receptor that is 20 exons long and produces two known protein isoforms differing in C-terminal amino acid composition, referred to as RET9 and RET51. Studies of human pheochromocytomas identified two additional novel transcripts involving the skipping of exon 3 or exons 3, 4, and 5 and are referred to as RET(Δ) (E3) and RET(Δ) (E345), respectively. Here we report the presence of Ret(Δ) (E3) and Ret(Δ) (E345) in zebrafish, mice, and rats and show that these transcripts are dynamically expressed throughout development of the CNS, peripheral nervous system, and kidneys. We further explore the biochemical properties of these isoforms, demonstrating that, like full-length RET, RET(ΔE3) and RET(ΔE345) are trafficked to the cell surface, interact with all four GFRα co-receptors, and have the ability to heterodimerize with full-length RET. Signaling experiments indicate that RET(ΔE3) is phosphorylated in a similar manner to full-length RET. RET(ΔE345), in contrast, displays higher baseline autophosphorylation, specifically on the catalytic tyrosine, Tyr(905), and also on one of the most important signaling residues, Tyr(1062) These data provide the first evidence for a physiologic role of these isoforms in RET pathway function. PMID:27226544

  6. Modulation of the tyrosine kinase receptor Ret/glial cell-derived neurotrophic factor (GDNF) signaling: a new player in reproduction induced anterior pituitary plasticity?

    PubMed

    Guillou, Anne; Romanò, Nicola; Bonnefont, Xavier; Le Tissier, Paul; Mollard, Patrice; Martin, Agnès O

    2011-02-01

    During gestation, parturition, and lactation, the endocrine axis of the dam must continually adapt to ensure the continual and healthy development of offspring. The anterior pituitary gland, which serves as the endocrine interface between the brain and periphery, undergoes adaptations that contribute to regulation of the reproductive axis. Growth factors and their receptors are potential candidates for intrapituitary and paracrine factors to participate in the functional and anatomical plasticity of the gland. We examined the involvement of the growth factor glial cell-derived neurotrophic factor (GDNF) and its receptor tyrosine kinase rearranged during transfection (Ret) in the physiological functional and anatomical plasticity of the anterior pituitary gland. We found that variations in both expression and subcellular localization of Ret during gestation and lactation are temporally correlated with changes in pituitary gland function. We showed that Ret/GDNF signaling could endorse two different functional roles depending on the physiological status. At the end of lactation and after weaning, Ret was colocalized with markers of apoptosis. We found that Ret could therefore act as a physiological dependence receptor capable of inducing apoptosis in the absence of GDNF. In addition, we identified the follicullostellate cell as a probable source for intrapituitary GDNF and proposed GDNF as a potential physiological modulator of endocrine cell function. During all stages studied, we showed that acute application of GDNF to pituitary slices was able to modulate both positively and negatively intracellular calcium activity. Altogether our results implicate Ret/GDNF as a potent pleiotropic factor able to influence pituitary physiology during a period of high plasticity. PMID:21239429

  7. Parkin cooperates with GDNF/RET signaling to prevent dopaminergic neuron degeneration

    PubMed Central

    Meka, Durga Praveen; Müller-Rischart, Anne Kathrin; Nidadavolu, Prakash; Mohammadi, Behnam; Motori, Elisa; Ponna, Srinivas Kumar; Aboutalebi, Helia; Bassal, Mahmoud; Annamneedi, Anil; Finckh, Barbara; Miesbauer, Margit; Rotermund, Natalie; Lohr, Christian; Tatzelt, Jörg; Winklhofer, Konstanze F.; Kramer, Edgar R.

    2015-01-01

    Parkin and the glial cell line–derived neurotrophic factor (GDNF) receptor RET have both been independently linked to the dopaminergic neuron degeneration that underlies Parkinson’s disease (PD). In the present study, we demonstrate that there is genetic crosstalk between parkin and the receptor tyrosine kinase RET in two different mouse models of PD. Mice lacking both parkin and RET exhibited accelerated dopaminergic cell and axonal loss compared with parkin-deficient animals, which showed none, and RET-deficient mice, in which we found moderate degeneration. Transgenic expression of parkin protected the dopaminergic systems of aged RET-deficient mice. Downregulation of either parkin or RET in neuronal cells impaired mitochondrial function and morphology. Parkin expression restored mitochondrial function in GDNF/RET-deficient cells, while GDNF stimulation rescued mitochondrial defects in parkin-deficient cells. In both cases, improved mitochondrial function was the result of activation of the prosurvival NF-κB pathway, which was mediated by RET through the phosphoinositide-3-kinase (PI3K) pathway. Taken together, these observations indicate that parkin and the RET signaling cascade converge to control mitochondrial integrity and thereby properly maintain substantia nigra pars compacta dopaminergic neurons and their innervation in the striatum. The demonstration of crosstalk between parkin and RET highlights the interplay in the protein network that is altered in PD and suggests potential therapeutic targets and strategies to treat PD. PMID:25822020

  8. 7-dehydrocholesterol efficiently supports Ret signaling in a mouse model of Smith-Opitz-Lemli syndrome

    PubMed Central

    Gou-Fàbregas, Myriam; Macià, Anna; Anerillas, Carlos; Vaquero, Marta; Jové, Mariona; Jain, Sanjay; Ribera, Joan; Encinas, Mario

    2016-01-01

    Smith-Lemli-Opitz syndrome (SLOS) is a rare disorder of cholesterol synthesis. Affected individuals exhibit growth failure, intellectual disability and a broad spectrum of developmental malformations. Among them, renal agenesis or hypoplasia, decreased innervation of the gut, and ptosis are consistent with impaired Ret signaling. Ret is a receptor tyrosine kinase that achieves full activity when recruited to lipid rafts. Mice mutant for Ret are born with no kidneys and enteric neurons, and display sympathetic nervous system defects causing ptosis. Since cholesterol is a critical component of lipid rafts, here we tested the hypothesis of whether the cause of the above malformations found in SLOS is defective Ret signaling owing to improper lipid raft composition or function. No defects consistent with decreased Ret signaling were found in newborn Dhcr7−/− mice, or in Dhcr7−/− mice lacking one copy of Ret. Although kidneys from Dhcr7−/− mice showed a mild branching defect in vitro, GDNF was able to support survival and downstream signaling of sympathetic neurons. Consistently, GFRα1 correctly partitioned to lipid rafts in brain tissue. Finally, replacement experiments demonstrated that 7-DHC efficiently supports Ret signaling in vitro. Taken together, our findings do not support a role of Ret signaling in the pathogenesis of SLOS. PMID:27334845

  9. Crosstalk between VEGF-A/VEGFR2 and GDNF/RET signaling pathways

    SciTech Connect

    Tufro, Alda . E-mail: atufro@aecom.yu.edu; Teichman, Jason; Banu, Nazifa; Villegas, Guillermo

    2007-06-29

    Vascular endothelial growth factor (VEGF-A) plays multiple roles in kidney development: stimulates cell proliferation, survival, tubulogenesis, and branching morphogenesis. However, the mechanism that mediates VEGF-A induced ureteric bud branching is unclear. Glial-derived neurotrophic factor (GDNF) signaling through tyrosine kinase c-RET is the major regulator of ureteric bud branching. Here we examined whether VEGF-A regulates RET signaling. We determined that ureteric bud-derived cells express the main VEGF-A signaling receptor, VEGFR2 and RET, by RT-PCR, immunoblotting, and immunocytochemistry. We show that the VEGF-A isoform VEGF{sub 165} induces RET-tyr{sup 1062} phosphorylation in addition to VEGFR2 autophosphorylation, that VEGF{sub 165} and GDNF have additive effects on RET-tyr{sup 1062} phosphorylation, and that VEGFR2 and RET co-immunoprecipitate. Functionally, VEGF{sub 165} induces ureteric bud cell proliferation and branching morphogenesis. Similarly, in embryonic kidney explants VEGF{sub 165} induces RET-tyr{sup 1062} phosphorylation and upregulates GDNF. These findings provide evidence for a novel cooperative interaction between VEGFR2 and RET that mediates VEGF-A functions in ureteric bud cells.

  10. A protein tyrosine kinase receptor, c-RET signaling pathway contributes to the enteric neurogenesis induced by a 5-HT4 receptor agonist at an anastomosis after transection of the gut in rodents.

    PubMed

    Goto, Kei; Kawahara, Isao; Kuniyasu, Hiroki; Takaki, Miyako

    2015-07-01

    We previously reported that a serotonin 4 (5-HT4) receptor agonist, mosapride citrate (MOS), increased the number of c-RET-positive cells and levels of c-RET mRNA in gel sponge implanted in the necks of rats. The 5-HT4 receptor is a G protein coupled receptor (GPCR) coupled to G protein Gs-cAMP cascades. We investigated the possibility that 5-HT4 receptor activation induced c-RET activation and/or PKA activation by elevating cAMP levels. Rodents were orally administered MOS by adding it to drinking water for 2 weeks after enteric nerve circuit insult via gut transection and anastomosis, together with the RET inhibitors withaferin A (WA) and RPI-1 or the PKA inhibitor H89. We then examined PGP9.5-positive cells in the newly formed granulation tissue at the anastomotic site. MOS significantly increased the number of new neurons, but not when co-administered with WA or RPI-1. Co-administration of H89 failed to alter MOS-induced increases in neurogenesis. In conclusion, the c-RET signaling pathway contributes to enteric neurogenesis facilitated by MOS, though the contribution of PKA activation seems unlikely. PMID:25850922

  11. Control of Aβ release from human neurons by differentiation status and RET signaling.

    PubMed

    Scholz, Diana; Chernyshova, Yana; Leist, Marcel

    2013-01-01

    Few studies have compared the processing of endogenous human amyloid precursor protein (APP) in younger and older neurons. Here, we characterized LUHMES cells as a human model to study Alzheimer's disease-related processes during neuronal maturation and aging. Differentiated LUHMES expressed and spontaneously processed APP via the secretase pathways, and they secreted amyloid β (Aβ) peptide. This was inhibited by cholesterol depletion or secretase inhibition, but not by block of tau phosphorylation. In vitro aged cells increased Aβ secretion without upregulation of APP or secretases. We identified the medium constituent glial cell line-derived neurotrophic factor (GDNF) as responsible for this effect. GDNF-triggered Aβ release was associated with rapid upregulation of the GDNF coreceptor "rearranged during transfection" (RET). Other direct (neurturin) or indirect (nerve growth factor) RET activators also increased Aβ, whereas different neurotrophins were ineffective. Downstream of RET, we found activation of protein kinase B (AKT) to be involved. Accordingly, inhibitors of the AKT regulator phosphatidylinositol-3-kinase completely blocked GDNF-triggered AKT phosphorylation and Aβ increase. This suggests that RET signaling affects Aβ release from aging neurons. PMID:22534065

  12. The β-catenin Axis Integrates Multiple Signals Downstream From RET/PTC and Leads to Cell Proliferation

    PubMed Central

    Castellone, Maria Domenica; De Falco, Valentina; Rao, Deva Magendra; Bellelli, Roberto; Muthu, Magesh; Basolo, Fulvio; Fusco, Alfredo; Gutkind, J. Silvio; Santoro, Massimo

    2009-01-01

    RET/PTC (RET/papillary thyroid carcinoma) oncoproteins result from the in-frame fusion of the RET receptor tyrosine kinase domain with protein dimerization motifs encoded by heterologous genes. Here we show that RET/PTC stimulates the β-catenin pathway. By stimulating PI3K/AKT and Ras/ERK, RET/PTC promotes GSK3β phosphorylation, thereby reducing GSK3β-mediated N-terminal β-catenin (Ser33/Ser37/Thr41) phosphorylation. In addition, RET/PTC physically interacts with β-catenin, and increases its phosphotyrosine content. The increased free pool of S/T(nonphospho)/Y(phospho)β-catenin is stabilized as a result of the reduced binding affinity for the Axin/GSK3β complex and activates the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor. Moreover, through the ERK pathway, RET/PTC stimulates cAMP-responsive element binding protein (CREB) phosphorylation and promotes the formation of a β-catenin-CREB-CBP/p300 transcriptional complex. Transcriptional complexes containing β-catenin are recruited to the cyclin D1 promoter and a cyclin D1 gene promoter reporter is active in RET/PTC expressing cells. Silencing of β-catenin by siRNA inhibits proliferation of RET/PTC transformed PC thyrocytes, whereas a constitutively active form of β-catenin stimulates autonomous proliferation of thyroid cells. Thus, multiple signaling events downstream from RET/PTC converge on β-catenin to stimulate cell proliferation. PMID:19223551

  13. The discovery of 2-substituted phenol quinazolines as potent RET kinase inhibitors with improved KDR selectivity.

    PubMed

    Newton, Rebecca; Bowler, Katherine A; Burns, Emily M; Chapman, Philip J; Fairweather, Emma E; Fritzl, Samantha J R; Goldberg, Kristin M; Hamilton, Niall M; Holt, Sarah V; Hopkins, Gemma V; Jones, Stuart D; Jordan, Allan M; Lyons, Amanda J; Nikki March, H; McDonald, Neil Q; Maguire, Laura A; Mould, Daniel P; Purkiss, Andrew G; Small, Helen F; Stowell, Alexandra I J; Thomson, Graeme J; Waddell, Ian D; Waszkowycz, Bohdan; Watson, Amanda J; Ogilvie, Donald J

    2016-04-13

    Deregulation of the receptor tyrosine kinase RET has been implicated in medullary thyroid cancer, a small percentage of lung adenocarcinomas, endocrine-resistant breast cancer and pancreatic cancer. There are several clinically approved multi-kinase inhibitors that target RET as a secondary pharmacology but additional activities, most notably inhibition of KDR, lead to dose-limiting toxicities. There is, therefore, a clinical need for more specific RET kinase inhibitors. Herein we report our efforts towards identifying a potent and selective RET inhibitor using vandetanib 1 as the starting point for structure-based drug design. Phenolic anilinoquinazolines exemplified by 6 showed improved affinities towards RET but, unsurprisingly, suffered from high metabolic clearance. Efforts to mitigate the metabolic liability of the phenol led to the discovery that a flanking substituent not only improved the hepatocyte stability, but could also impart a significant gain in selectivity. This culminated in the identification of 36; a potent RET inhibitor with much improved selectivity against KDR. PMID:26874741

  14. Characterization of interactions and pharmacophore development for DFG-out inhibitors to RET tyrosine kinase.

    PubMed

    Gao, Chunxia; Grøtli, Morten; Eriksson, Leif A

    2015-07-01

    RET (rearranged during transfection) tyrosine kinase is a promising target for several human cancers. Abt-348, Birb-796, Motesanib and Sorafenib are DFG-out multi-kinase inhibitors that have been reported to inhibit RET activity with good IC50 values. Although the DFG-out conformation has attracted great interest in the design of type II inhibitors, the structural requirements for binding to the RET DFG-out conformation remains unclear. Herein, the DFG-out conformation of RET was determined by homology modelling, the four inhibitors were docked, and the binding modes investigated by molecular dynamics simulation. Binding free energies were calculated using the molecular mechanics/Poisson-Bolzmann surface area (MM/PBSA) method. The trends in predicted binding free affinities correlated well with experimental data and were used to explain the activity difference of the studied inhibitors. Per-residue energy decomposition analyses provided further information on specific interaction properties. Finally, we also conducted a detailed e-pharmacophore modelling of the different RET-inhibitor complexes, explaining the common and specific pharmacophore features of the different complexes. The results reported herein will be useful in future rational design of novel DFG-out RET inhibitors. PMID:26044359

  15. Traditional and targeted exome sequencing reveals common, rare and novel functional deleterious variants in RET signaling complex in a cohort of living US patients with urinary tract malformations

    PubMed Central

    Chatterjee, Rajshekhar; Ramos, Enrique; Hoffman, Mary; VanWinkle, Jessica; Martin, Daniel R; Davis, Thomas K; Hoshi, Masato; Hmiel, Stanley P; Beck, Anne; Hruska, Keith; Coplen, Doug; Liapis, Helen; Mitra, Robi; Druley, Todd; Austin, Paul; Jain, Sanjay

    2013-01-01

    Signaling by the glial cell line-derived neurotrophic factor (GDNF)-RET receptor tyrosine kinase and SPRY1, a RET repressor, is essential for early urinary tract development. Individual or a combination of GDNF, RET and SPRY1 mutant alleles in mice cause renal malformations reminiscent of congenital anomalies of the kidney or urinary tract (CAKUT) in humans and distinct from renal agenesis phenotype in complete GDNF or RET null mice. We sequenced GDNF, SPRY1 and RET in 122 unrelated living CAKUT patients to discover deleterious mutations that cause CAKUT. Novel or rare deleterious mutations in GDNF or RET were found in 6 unrelated patients. A family with duplicated collecting system had a novel mutation, RETR831Q, which showed markedly decreased GDNF dependent MAPK activity. Two patients with RET-G691S polymorphism harbored additional rare non-synonymous variants GDNF-R93W and RET-R982C. The patient with double RET-G691S/R982C genotype had multiple defects including renal dysplasia, megaureters and cryptorchidism. Presence of both mutations were necessary to affect RET activity. Targeted whole exome and next-generation sequencing revealed a novel deleterious mutation G443D in GFRα1, the co-receptor for RET, in this patient. Pedigree analysis indicated that the GFRα1 mutation was inherited from the unaffected mother and the RET mutations from the unaffected father. Our studies indicate that 5% of living CAKUT patients harbor deleterious rare variants or novel mutations in GDNF-GFRα1-RET pathway. We provide evidence for the coexistence of deleterious rare and common variants in genes in the same pathway as a cause of CAKUT and discovered novel phenotypes associated with the RET pathway. PMID:22729463

  16. Identification of two novel RET kinase inhibitors through MCR-based drug discovery: Design, synthesis and evaluation

    PubMed Central

    Frett, Brendan; Moccia, Marialuisa; Carlomagno, Francesca; Santoro, Massimo; Li, Hong-yu

    2015-01-01

    From an MCR fragment library, two novel chemical series have been developed as inhibitors of RET, which is a kinase involved in the pathology of medullary thyroid cancer (MTC). Structure activity relationship studies (SAR) identified two sub-micromolar tractable leads, 6g and 13g. 6g was confirmed to be a Type-II RET inhibitor. 13g and 6g inhibited RET in cells transformed by RET/C634. A RET DFG-out homology model was established and utilized to predict Type-II inhibitor binding modes. PMID:25232968

  17. Congenital central hypoventilation syndrome: Mutation analysis of the receptor tyrosine kinase RET

    SciTech Connect

    Bolk, S.; Angrist, M.; Schwartz, S.; Chakravarti, A.

    1996-06-28

    Congenital central hypoventilation syndrome (CCHS) usually occurs as an isolated phenotype. However, 16% of the index cases are also affected with Hirschsprung disease (HSCR). Complex segregation analysis suggests that CCHS is familial and has the same inheritance pattern with or without HSCR. We postulate that alteration of normal function of the receptor tyrosine kinase, RET, may contribute to CCHS based on RET`s expression pattern and the identification of RET mutations in HSCR patients. To further explore the nature of the inheritance of CCHS, we have undertaken two main routes of investigation: cytogenetic analysis and mutation detection. Cytogenetic analysis of metaphase chromosomes showed normal karyotypes in 13 of the 14 evaluated index cases; one index case carried a familial pericentric inversion on chromosome 2. Mutation analysis showed no sequence changes unique to index cases, as compared to control individuals, and as studied by single strand conformational polymorphism (SSCP) analysis of the coding region of RET. We conclude that point mutations in the RET coding region cannot account for a substantial fraction of CCHS in this patient population, and that other candidate genes involved in neural crest cell differentiation and development must be considered. 54 refs.

  18. RET/GFRα Signals Are Dispensable for Thymic T Cell Development In Vivo

    PubMed Central

    Almeida, Afonso Rocha Martins; Arroz-Madeira, Sílvia; Fonseca-Pereira, Diogo; Ribeiro, Hélder; Lasrado, Reena; Pachnis, Vassilis; Veiga-Fernandes, Henrique

    2012-01-01

    Identification of thymocyte regulators is a central issue in T cell biology. Interestingly, growing evidence indicates that common key molecules control neuronal and immune cell functions. The neurotrophic factor receptor RET mediates critical functions in foetal hematopoietic subsets, thus raising the possibility that RET-related molecules may also control T cell development. We show that Ret, Gfra1 and Gfra2 are abundantly expressed by foetal and adult immature DN thymocytes. Despite the developmentally regulated expression of these genes, analysis of foetal thymi from Gfra1, Gfra2 or Ret deficient embryos revealed that these molecules are dispensable for foetal T cell development. Furthermore, analysis of RET gain of function and Ret conditional knockout mice showed that RET is also unnecessary for adult thymopoiesis. Finally, competitive thymic reconstitution assays indicated that Ret deficient thymocytes maintained their differentiation fitness even in stringent developmental conditions. Thus, our data demonstrate that RET/GFRα signals are dispensable for thymic T cell development in vivo, indicating that pharmacological targeting of RET signalling in tumours is not likely to result in T cell production failure. PMID:23300832

  19. Drosophila Cancer Models Identify Functional Differences between Ret Fusions.

    PubMed

    Levinson, Sarah; Cagan, Ross L

    2016-09-13

    We generated and compared Drosophila models of RET fusions CCDC6-RET and NCOA4-RET. Both RET fusions directed cells to migrate, delaminate, and undergo EMT, and both resulted in lethality when broadly expressed. In all phenotypes examined, NCOA4-RET was more severe than CCDC6-RET, mirroring their effects on patients. A functional screen against the Drosophila kinome and a library of cancer drugs found that CCDC6-RET and NCOA4-RET acted through different signaling networks and displayed distinct drug sensitivities. Combining data from the kinome and drug screens identified the WEE1 inhibitor AZD1775 plus the multi-kinase inhibitor sorafenib as a synergistic drug combination that is specific for NCOA4-RET. Our work emphasizes the importance of identifying and tailoring a patient's treatment to their specific RET fusion isoform and identifies a multi-targeted therapy that may prove effective against tumors containing the NCOA4-RET fusion. PMID:27626672

  20. A Caged Ret Kinase Inhibitor and its Effect on Motoneuron Development in Zebrafish Embryos

    PubMed Central

    Bliman, David; Nilsson, Jesper R.; Kettunen, Petronella; Andréasson, Joakim; Grøtli, Morten

    2015-01-01

    Proto-oncogene tyrosine-protein kinase receptor RET is implicated in the development and maintenance of neurons of the central and peripheral nervous systems. Attaching activity-compromising photocleavable groups (caging) to inhibitors could allow for external spatiotemporally controlled inhibition using light, potentially providing novel information on how these kinase receptors are involved in cellular processes. Here, caged RET inhibitors were obtained from 3-substituted pyrazolopyrimidine-based compounds by attaching photolabile groups to the exocyclic amino function. The most promising compound displayed excellent inhibitory effect in cell-free, as well as live-cell assays upon decaging. Furthermore, inhibition could be efficiently activated with light in vivo in zebrafish embryos and was shown to effect motoneuron development. PMID:26300345

  1. Anilinoquinazoline inhibitors of the RET kinase domain-Elaboration of the 7-position.

    PubMed

    Jordan, Allan M; Begum, Habiba; Fairweather, Emma; Fritzl, Samantha; Goldberg, Kristin; Hopkins, Gemma V; Hamilton, Niall M; Lyons, Amanda J; March, H Nikki; Newton, Rebecca; Small, Helen F; Vishwanath, Swamy; Waddell, Ian D; Waszkowycz, Bohdan; Watson, Amanda J; Ogilvie, Donald J

    2016-06-01

    We have previously reported a series of anilinoquinazoline derivatives as potent and selective biochemical inhibitors of the RET kinase domain. However, these derivatives displayed diminished cellular potency. Herein we describe further optimisation of the series through modification of their physicochemical properties, delivering improvements in cell potency. However, whilst cellular selectivity against key targets could be maintained, combining cell potency and acceptable pharmacokinetics proved challenging. PMID:27086121

  2. Alternative splicing results in RET isoforms with distinct trafficking properties

    PubMed Central

    Richardson, Douglas S.; Rodrigues, David M.; Hyndman, Brandy D.; Crupi, Mathieu J. F.; Nicolescu, Adrian C.; Mulligan, Lois M.

    2012-01-01

    RET encodes a receptor tyrosine kinase that is essential for spermatogenesis, development of the sensory, sympathetic, parasympathetic, and enteric nervous systems and the kidneys, as well as for maintenance of adult midbrain dopaminergic neurons. RET is alternatively spliced to encode multiple isoforms that differ in their C-terminal amino acids. The RET9 and RET51 isoforms display unique levels of autophosphorylation and have differential interactions with adaptor proteins. They induce distinct gene expression patterns, promote different levels of cell differentiation and transformation, and play unique roles in development. Here we present a comprehensive study of the subcellular localization and trafficking of RET isoforms. We show that immature RET9 accumulates intracellularly in the Golgi, whereas RET51 is efficiently matured and present in relatively higher amounts on the plasma membrane. RET51 is internalized faster after ligand binding and undergoes recycling back to the plasma membrane. This differential trafficking of RET isoforms produces a more rapid and longer duration of signaling through the extracellular-signal regulated kinase/mitogen-activated protein kinase pathway downstream of RET51 relative to RET9. Together these differences in trafficking properties contribute to some of the functional differences previously observed between RET9 and RET51 and establish the important role of intracellular trafficking in modulating and maintaining RET signaling. PMID:22875993

  3. GFRA2 Identifies Cardiac Progenitors and Mediates Cardiomyocyte Differentiation in a RET-Independent Signaling Pathway.

    PubMed

    Ishida, Hidekazu; Saba, Rie; Kokkinopoulos, Ioannis; Hashimoto, Masakazu; Yamaguchi, Osamu; Nowotschin, Sonja; Shiraishi, Manabu; Ruchaya, Prashant; Miller, Duncan; Harmer, Stephen; Poliandri, Ariel; Kogaki, Shigetoyo; Sakata, Yasushi; Dunkel, Leo; Tinker, Andrew; Hadjantonakis, Anna-Katerina; Sawa, Yoshiki; Sasaki, Hiroshi; Ozono, Keiichi; Suzuki, Ken; Yashiro, Kenta

    2016-07-26

    A surface marker that distinctly identifies cardiac progenitors (CPs) is essential for the robust isolation of these cells, circumventing the necessity of genetic modification. Here, we demonstrate that a Glycosylphosphatidylinositol-anchor containing neurotrophic factor receptor, Glial cell line-derived neurotrophic factor receptor alpha 2 (Gfra2), specifically marks CPs. GFRA2 expression facilitates the isolation of CPs by fluorescence activated cell sorting from differentiating mouse and human pluripotent stem cells. Gfra2 mutants reveal an important role for GFRA2 in cardiomyocyte differentiation and development both in vitro and in vivo. Mechanistically, the cardiac GFRA2 signaling pathway is distinct from the canonical pathway dependent on the RET tyrosine kinase and its established ligands. Collectively, our findings establish a platform for investigating the biology of CPs as a foundation for future development of CP transplantation for treating heart failure. PMID:27396331

  4. GDNF-Ret signaling in midbrain dopaminergic neurons and its implication for Parkinson disease.

    PubMed

    Kramer, Edgar R; Liss, Birgit

    2015-12-21

    Glial cell line-derived neurotrophic factor (GDNF) and its canonical receptor Ret can signal together or independently to fulfill many important functions in the midbrain dopaminergic (DA) system. While Ret signaling clearly impacts on the development, maintenance and regeneration of the mesostriatal DA system, the physiological functions of GDNF for the DA system are still unclear. Nevertheless, GDNF is still considered to be an excellent candidate to protect and/or regenerate the mesostriatal DA system in Parkinson disease (PD). Clinical trials with GDNF on PD patients are, however, so far inconclusive. Here, we review the current knowledge of GDNF and Ret signaling and function in the midbrain DA system, and their crosstalk with proteins and signaling pathways associated with PD. PMID:26555190

  5. Intracellular Signal Transduction and Modification of the Tumor Microenvironment Induced by RET/PTCs in Papillary Thyroid Carcinoma

    PubMed Central

    Menicali, Elisa; Moretti, Sonia; Voce, Pasquale; Romagnoli, Serena; Avenia, Nicola; Puxeddu, Efisio

    2012-01-01

    RET gene rearrangements (RET/PTCs) represent together with BRAF point mutations the two major groups of mutations involved in papillary thyroid carcinoma (PTC) initiation and progression. In this review, we will examine the mechanisms involved in RET/PTC-induced thyroid cell transformation. In detail, we will summarize the data on the molecular mechanisms involved in RET/PTC formation and in its function as a dominant oncogene, on the activated signal transduction pathways and on the induced gene expression modifications. Moreover, we will report on the effects of RET/PTCs on the tumor microenvironment. Finally, a short review of the literature on RET/PTC prognostic significance will be presented. PMID:22661970

  6. An orally available tyrosine kinase ALK and RET dual inhibitor bearing the tetracyclic benzo[b]carbazolone core.

    PubMed

    Song, Zilan; Xia, Zongjun; Ji, Yinchun; Xing, Li; Gao, Yinglei; Ai, Jing; Geng, Meiyu; Zhang, Ao

    2016-08-01

    Our early structure-activity relationship study has identified benzo[b]carbazolone 6 as a high potency orally bioavailable ALK inhibitor. Further lead profiling disclosed that 6 is active against both ALK resistant and hot spot-activating mutants, and is also highly potent against RET kinase. Tumor stasis and partial tumor regression were achieved with 6 in both NIH/3T3-EML4-ALK and NIH/3T3-EML4-ALK L1196M xenograft models. Based on the optimal in vitro and in vivo antitumor efficacy, compound 6 is now being profiled further in our preclinical settings as a new orally available ALK/RET dual inhibitor. PMID:27131066

  7. Distinct Temporal Regulation of RET Isoform Internalization: Roles of Clathrin and AP2.

    PubMed

    Crupi, Mathieu J F; Yoganathan, Piriya; Bone, Leslie N; Lian, Eric; Fetz, Andrew; Antonescu, Costin N; Mulligan, Lois M

    2015-11-01

    The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers. RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes. RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line-derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 μ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions. PMID:26304132

  8. The histone code reader SPIN1 controls RET signaling in liposarcoma

    PubMed Central

    Franz, Henriette; Greschik, Holger; Willmann, Dominica; Ozretić, Luka; Jilg, Cordula Annette; Wardelmann, Eva; Jung, Manfred; Buettner, Reinhard; Schüle, Roland

    2015-01-01

    The histone code reader Spindlin1 (SPIN1) has been implicated in tumorigenesis and tumor growth, but the underlying molecular mechanisms remain poorly understood. Here, we show that reducing SPIN1 levels strongly impairs proliferation and increases apoptosis of liposarcoma cells in vitro and in xenograft mouse models. Combining signaling pathway, genome-wide chromatin binding, and transcriptome analyses, we found that SPIN1 directly enhances expression of GDNF, an activator of the RET signaling pathway, in cooperation with the transcription factor MAZ. Accordingly, knockdown of SPIN1 or MAZ results in reduced levels of GDNF and activated RET explaining diminished liposarcoma cell proliferation and survival. In line with these observations, levels of SPIN1, GDNF, activated RET, and MAZ are increased in human liposarcoma compared to normal adipose tissue or lipoma. Importantly, a mutation of SPIN1 within the reader domain interfering with chromatin binding reduces liposarcoma cell proliferation and survival. Together, our data describe a molecular mechanism for SPIN1 function in liposarcoma and suggest that targeting SPIN1 chromatin association with small molecule inhibitors may represent a novel therapeutic strategy. PMID:25749382

  9. The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells

    PubMed Central

    Melillo, Rosa Marina; Castellone, Maria Domenica; Guarino, Valentina; De Falco, Valentina; Cirafici, Anna Maria; Salvatore, Giuliana; Caiazzo, Fiorina; Basolo, Fulvio; Giannini, Riccardo; Kruhoffer, Mogens; Orntoft, Torben; Fusco, Alfredo; Santoro, Massimo

    2005-01-01

    In papillary thyroid carcinomas (PTCs), rearrangements of the RET receptor (RET/PTC) and activating mutations in the BRAF or RAS oncogenes are mutually exclusive. Here we show that the 3 proteins function along a linear oncogenic signaling cascade in which RET/PTC induces RAS-dependent BRAF activation and RAS- and BRAF-dependent ERK activation. Adoptive activation of the RET/PTC-RAS-BRAF axis induced cell proliferation and Matrigel invasion of thyroid follicular cells. Gene expression profiling revealed that the 3 oncogenes activate a common transcriptional program in thyroid cells that includes upregulation of the CXCL1 and CXCL10 chemokines, which in turn stimulate proliferation and invasion. Thus, motile and mitogenic properties are intrinsic to transformed thyroid cells and are governed by an epistatic oncogenic signaling cascade. PMID:15761501

  10. Preparation of 3-substituted-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amines as RET kinase inhibitors.

    PubMed

    Dinér, Peter; Alao, John P; Söderlund, Johan; Sunnerhagen, Per; Grøtli, Morten

    2012-05-24

    A series of 3-substituted-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amines have been designed, synthesized, and evaluated as RET protein kinase inhibitors. On the basis of docking results, a small library of pyrazolopyrimidine compounds with an extended hydrophobic side arm was synthesized. The most promising of the compounds (7a) displayed efficient inhibition in vitro and good selectivity when tested on a panel of kinases. Furthermore, 7a inhibited GDNF-induced RET phosphorylation of ERK1/2 in MCF-7 breast cancer cells at concentrations as low as 100 nM. PMID:22559926

  11. Computational Modeling of Structurally Conserved Cancer Mutations in the RET and MET Kinases: The Impact on Protein Structure, Dynamics, and Stability

    PubMed Central

    Dixit, Anshuman; Torkamani, Ali; Schork, Nicholas J.; Verkhivker, Gennady

    2009-01-01

    Structural and biochemical characterization of protein kinases that confer oncogene addiction and harbor a large number of disease-associated mutations, including RET and MET kinases, have provided insights into molecular mechanisms associated with the protein kinase activation in human cancer. In this article, structural modeling, molecular dynamics, and free energy simulations of a structurally conserved mutational hotspot, shared by M918T in RET and M1250T in MET kinases, are undertaken to quantify the molecular mechanism of activation and the functional role of cancer mutations in altering protein kinase structure, dynamics, and stability. The mechanistic basis of the activating RET and MET cancer mutations may be driven by an appreciable free energy destabilization of the inactive kinase state in the mutational forms. According to our results, the locally enhanced mobility of the cancer mutants and a higher conformational entropy are counterbalanced by a larger enthalpy loss and result in the decreased thermodynamic stability. The computed protein stability differences between the wild-type and cancer kinase mutants are consistent with circular dichroism spectroscopy and differential scanning calorimetry experiments. These results support the molecular mechanism of activation, which causes a detrimental imbalance in the dynamic equilibrium shifted toward the active form of the enzyme. Furthermore, computer simulations of the inhibitor binding with the oncogenic and drug-resistant RET mutations have also provided a plausible molecular rationale for the observed differences in the inhibition profiles, which is consistent with the experimental data. Finally, structural mapping of RET and MET cancer mutations and the computed protein stability changes suggest a similar mechanism of activation, whereby the cancer mutations which display the higher oncogenic activity tend to have the greatest destabilization effect on the inactive kinase structure. PMID:19186126

  12. MAP kinase cascades: scaffolding signal specificity.

    PubMed

    van Drogen, Frank; Peter, Matthias

    2002-01-22

    Scaffold proteins organize many MAP kinase pathways by interacting with several components of these cascades. Recent studies suggest that scaffold proteins provide local activation platforms that contribute to signal specificity by insulating different MAP kinase pathways. PMID:11818078

  13. Targeting mTOR in RET mutant medullary and differentiated thyroid cancer cells

    PubMed Central

    Gild, Matti L; Landa, Iñigo; Ryder, Mabel; Ghossein, Ronald A; Knauf, Jeffrey A; Fagin, James A

    2015-01-01

    Inhibitors of RET, a tyrosine kinase receptor encoded by a gene that is frequently mutated in medullary thyroid cancer, have emerged as promising novel therapies for the disease. Rapalogs and other mammalian target of rapamycin (mTOR) inhibitors are effective agents in patients with gastroenteropancreatic neuroendocrine tumors, which share lineage properties with medullary thyroid carcinomas. The objective of this study was to investigate the contribution of mTOR activity to RET-induced signaling and cell growth and to establish whether growth suppression is enhanced by co-targeting RET and mTOR kinase activities. Treatment of the RET mutant cell lines TT, TPC-1, and MZ-CRC-1 with AST487, a RET kinase inhibitor, suppressed growth and showed profound and sustained inhibition of mTOR signaling, which was recapitulated by siRNA-mediated RET knockdown. Inhibition of mTOR with INK128, a dual mTORC1 and mTORC2 kinase inhibitor, also resulted in marked growth suppression to levels similar to those seen with RET blockade. Moreover, combined treatment with AST487 and INK128 at low concentrations suppressed growth and induced apoptosis. These data establish mTOR as a key mediator of RET-mediated cell growth in thyroid cancer cells and provide a rationale for combinatorial treatments in thyroid cancers with oncogenic RET mutations. PMID:23828865

  14. Identification of a lung adenocarcinoma cell line with CCDC6-RET fusion gene and the effect of RET inhibitors in vitro and in vivo.

    PubMed

    Suzuki, Makito; Makinoshima, Hideki; Matsumoto, Shingo; Suzuki, Ayako; Mimaki, Sachiyo; Matsushima, Koutatsu; Yoh, Kiyotaka; Goto, Koichi; Suzuki, Yutaka; Ishii, Genichiro; Ochiai, Atsushi; Tsuta, Koji; Shibata, Tatsuhiro; Kohno, Takashi; Esumi, Hiroyasu; Tsuchihara, Katsuya

    2013-07-01

    Rearrangements of the proto-oncogene RET are newly identified potential driver mutations in lung adenocarcinoma (LAD). However, the absence of cell lines harboring RET fusion genes has hampered the investigation of the biological relevance of RET and the development of RET-targeted therapy. Thus, we aimed to identify a RET fusion positive LAD cell line. Eleven LAD cell lines were screened for RET fusion transcripts by reverse transcription-polymerase chain reaction. The biological relevance of the CCDC6-RET gene products was assessed by cell growth, survival and phosphorylation of ERK1/2 and AKT with or without the suppression of RET expression using RNA interference. The efficacy of RET inhibitors was evaluated in vitro using a culture system and in an in vivo xenograft model. Expression of the CCDC6-RET fusion gene in LC-2/ad cells was demonstrated by the mRNA and protein levels, and the genomic break-point was confirmed by genomic DNA sequencing. Mutations in KRAS and EGFR were not observed in the LC-2/ad cells. CCDC6-RET was constitutively active, and the introduction of a siRNA targeting the RET 3' region decreased cell proliferation by downregulating RET and ERK1/2 phosphorylation. Moreover, treatment with RET-inhibitors, including vandetanib, reduced cell viability, which was accompanied by the downregulation of the AKT and ERK1/2 signaling pathways. Vandetanib exhibited anti-tumor effects in the xenograft model. Endogenously expressing CCDC6-RET contributed to cell growth. The inhibition of kinase activity could be an effective treatment strategy for LAD. LC-2/ad is a useful model for developing fusion RET-targeted therapy. PMID:23578175

  15. High Affinity Pharmacological Profiling of Dual Inhibitors Targeting RET and VEGFR2 in Inhibition of Kinase and Angiogeneis Events in Medullary Thyroid Carcinoma.

    PubMed

    Dunna, Nageswara Rao; Kandula, Venkatesh; Girdhar, Amandeep; Pudutha, Amareshwari; Hussain, Tajamul; Bandaru, Srinivas; Nayarisseri, Anuraj

    2015-01-01

    Clinical evidence shows that dual inhibition of kinases as well angiogenesis provides ideal therapeutic option in the treatment of medullary thyroid carcinoma (MTC) than inhibiting either of these with the events separately. Although treatment with dual inhibitors has shown good clinical responses in patients with MTC, it has been associated with serious side effects. Some inhibitors are active agents for both angiogenesis or kinase activity. Owing to narrow therapeutic window of established inhibitors, the present study aims to identify high affinity dual inhibitors targeting RET and VEGFR2 respectively for kinase and angiogenesis activity. Established inhibitors like Vandetanib, Cabozantinib, Motesanib, PP121, RAF265 and Sunitinib served as query parent compounds for identification of structurally similar compounds by Tanimoto-based similarity searching with a threshold of 95% against the PubChem database. All the parent inhibitors and respective similar compounds were docked against RET and VEGFR2 in order to retrieve high affinity compounds with these two proteins. AGN-PC-0CUK9P PubCID: 59320403 a compound related to PPI21 showed almost equal affinity for RET and VEGFR2 and unlike other screened compounds with no apparent bias for either of the receptors. Further, AGN- PC-0CUK9P demonstrated appreciable interaction with both RET and VEGFR2 and superior kinase activity in addition to showed optimal ADMET properties and pharmacophore features. From our in silico investigation we suggest AGN-PC-0CUK9P as a superior dual inhibitor targeting RET and VEGFR2 with high efficacy which should be proposed for pharmacodynamic and pharmacokinetic studies for improved treatment of MTC. PMID:26514495

  16. KIF26A is an unconventional kinesin and regulates GDNF-Ret signaling in enteric neuronal development.

    PubMed

    Zhou, Ruyun; Niwa, Shinsuke; Homma, Noriko; Takei, Yosuke; Hirokawa, Nobutaka

    2009-11-13

    The kinesin superfamily proteins (KIFs) are motor proteins that transport organelles and protein complexes in a microtubule- and ATP-dependent manner. We identified KIF26A as a new member of the murine KIFs. KIF26A is a rather atypical member as it lacks ATPase activity. Mice with a homozygous deletion of Kif26a developed a megacolon with enteric nerve hyperplasia. Kif26a-/- enteric neurons showed hypersensitivity for GDNF-Ret signaling, and we find that KIF26A suppressed GDNF-Ret signaling by direct binding and inhibition of Grb2, an essential component of GDNF/Akt/ERK signaling. We therefore propose that the unconventional kinesin KIF26A plays a key role in enteric nervous system development by repressing a cell growth signaling pathway. PMID:19914172

  17. The Hybrid Histidine Kinase LadS Forms a Multicomponent Signal Transduction System with the GacS/GacA Two-Component System in Pseudomonas aeruginosa

    PubMed Central

    Redelberger, David; Fadel, Firas; Filloux, Alain; Sivaneson, Melissa; de Bentzmann, Sophie; Bordi, Christophe

    2016-01-01

    In response to environmental changes, Pseudomonas aeruginosa is able to switch from a planktonic (free swimming) to a sessile (biofilm) lifestyle. The two-component system (TCS) GacS/GacA activates the production of two small non-coding RNAs, RsmY and RsmZ, but four histidine kinases (HKs), RetS, GacS, LadS and PA1611, are instrumental in this process. RetS hybrid HK blocks GacS unorthodox HK autophosphorylation through the formation of a heterodimer. PA1611 hybrid HK, which is structurally related to GacS, interacts with RetS in P. aeruginosa in a very similar manner to GacS. LadS hybrid HK phenotypically antagonizes the function of RetS by a mechanism that has never been investigated. The four sensors are found in most Pseudomonas species but their characteristics and mode of signaling may differ from one species to another. Here, we demonstrated in P. aeruginosa that LadS controls both rsmY and rsmZ gene expression and that this regulation occurs through the GacS/GacA TCS. We additionally evidenced that in contrast to RetS, LadS signals through GacS/GacA without forming heterodimers, either with GacS or with RetS. Instead, we demonstrated that LadS is involved in a genuine phosphorelay, which requires both transmitter and receiver LadS domains. LadS signaling ultimately requires the alternative histidine-phosphotransfer domain of GacS, which is here used as an Hpt relay by the hybrid kinase. LadS HK thus forms, with the GacS/GacA TCS, a multicomponent signal transduction system with an original phosphorelay cascade, i.e. H1LadS→D1LadS→H2GacS→D2GacA. This highlights an original strategy in which a unique output, i.e. the modulation of sRNA levels, is controlled by a complex multi-sensing network to fine-tune an adapted biofilm and virulence response. PMID:27176226

  18. Kinase signaling in the spindle checkpoint.

    PubMed

    Kang, Jungseog; Yu, Hongtao

    2009-06-01

    The spindle checkpoint is a cell cycle surveillance system that ensures the fidelity of chromosome segregation. In mitosis, it elicits the "wait anaphase" signal to inhibit the anaphase-promoting complex or cyclosome until all chromosomes achieve bipolar microtubule attachment and align at the metaphase plate. Because a single kinetochore unattached to microtubules activates the checkpoint, the wait anaphase signal is thought to be generated by this kinetochore and is then amplified and distributed throughout the cell to inhibit the anaphase-promoting complex/cyclosome. Several spindle checkpoint kinases participate in the generation and amplification of this signal. Recent studies have begun to reveal the activation mechanisms of these checkpoint kinases. Increasing evidence also indicates that the checkpoint kinases not only help to generate the wait anaphase signal but also actively correct kinetochore-microtubule attachment defects. PMID:19228686

  19. Effect of retS gene on antibiotics production in Pseudomonas fluorescens FD6.

    PubMed

    Zhang, Qingxia; Xiao, Qi; Xu, Jingyou; Tong, Yunhui; Wen, Jia; Chen, Xijun; Wei, Lihui

    2015-11-01

    A hybrid sensor kinase termed RetS (regulator of exopolysaccharide and Type III secretion) controls expression of numerous genes in Pseudomonas aeruginosa. To investigate the function of RetS in P. fluorescens FD6, the retS gene was disrupted. Genetic inactivation of retS resulted in enhanced production of 2, 4-diacetylphloroglucinol, pyrrolnitrin, and pyoluteorin. The retS mutant also exhibited significant increase in phlA-lacZ, prnA-lacZ, and pltA-lacZ transcription levels, influencing expression levels of the small regulatory RNAs RsmX and RsmZ. In the gacSretS double mutant, all the phenotypic changes caused by the retS deletion were reversed to the level of gacS single mutant. Furthermore, the retS mutation drastically elevated biofilm formation and improved the colonization ability of strain FD6 on wheat rhizospheres. Based on these results, we proposed that RetS negatively controlled the production of antibiotics through the Gac/Rsm pathway in P. fluorescens FD6. PMID:26505308

  20. Osmotic stress signaling via protein kinases.

    PubMed

    Fujii, Hiroaki; Zhu, Jian-Kang

    2012-10-01

    Plants face various kinds of environmental stresses, including drought, salinity, and low temperature, which cause osmotic stress. An understanding of the plant signaling pathways that respond to osmotic stress is important for both basic biology and agriculture. In this review, we summarize recent investigations concerning the SNF1-related protein kinase (SnRK) 2 kinase family, which play central roles in osmotic stress responses. SnRK2s are activated by osmotic stress, and a mutant lacking SnRK2s is hypersensitive to osmotic stress. Many questions remain about the signaling pathway upstream and downstream of SnRK2s. Because some SnRK2s also functions in the abscisic acid (ABA) signaling pathway, which has recently been well clarified, study of SnRK2s in ABA signaling can provide clues regarding their roles in osmotic stress signaling. PMID:22828864

  1. To Bud or not to Bud: The RET perspective in CAKUT

    PubMed Central

    Davis, T. Keefe; Hoshi, Masato; Jain, Sanjay

    2013-01-01

    Congenital anomalies of the kidneys or lower urinary tract (CAKUT) encompass a spectrum of anomalies that result from aberrations in spatio-temporal regulation of genetic, epigenetic, environmental and molecular signals at key stages of urinary tract development. The Rearranged in Transfection (RET) tyrosine kinase signaling system is a major pathway required for normal development of the kidneys, ureters, peripheral and enteric nervous systems. In the kidneys, RET is activated by interaction with the ligand glial cell line-derived neurotrophic factor (GDNF) and coreceptor GFRα1. This activated complex regulates a number of downstream signaling cascades (PLCγ, MAPK and PI3K) that control proliferation, migration, renewal and apoptosis. Disruption of these events is thought to underlie diseases arising from aberrant RET signaling. RET mutations are found in 5–30% of CAKUT patients and a number of Ret mouse mutants show a spectrum of kidney and lower urinary tract defects reminiscent of CAKUT in humans. The remarkable similarities between mouse and human kidney development and in defects due to RET mutations has led to using RET signaling as a paradigm to determine the fundamental principles in patterning of the upper and lower urinary tract and to understand CAKUT pathogenesis. In this review we provide an overview of studies in vivo that delineate expression and the functional importance of RET signaling complex during different stages of development of the upper and lower urinary tracts. We discuss how RET signaling balances activating and inhibitory signals emanating from its docking tyrosines and its interaction with upstream and downstream regulators to precisely modulate different aspects of Wolffian duct patterning and branching morphogenesis. We outline the diversity of cellular mechanisms regulated by RET, disruption of which causes malformations ranging from renal agenesis to multicystic dysplastic kidneys in the upper tract and vesicoureteral reflux

  2. RET/PTC Translocations and Clinico-Pathological Features in Human Papillary Thyroid Carcinoma

    PubMed Central

    Romei, Cristina; Elisei, Rossella

    2012-01-01

    Thyroid carcinoma is the most frequent endocrine cancer accounting for 5–10% of thyroid nodules. Papillary histotype (PTC) is the most prevalent form accounting for 80% of all thyroid carcinoma. Although much is known about its epidemiology, pathogenesis, clinical, and biological behavior, the only documented risk factor for PTC is the ionizing radiation exposure. Rearrangements of the Rearranged during Transfection (RET) proto-oncogene are found in PTC and have been shown to play a pathogenic role. The first RET rearrangement, named RET/PTC, was discovered in 1987. This rearrangement constitutively activates the transcription of the RET tyrosine-kinase domain in follicular cell, thus triggering the signaling along the MAPK pathway and an uncontrolled proliferation. Up to now, 13 different types of RET/PTC rearrangements have been reported but the two most common are RET/PTC1 and RET/PTC3. Ionizing radiations are responsible for the generation of RET/PTC rearrangements, as supported by in vitro studies and by the evidence that RET/PTC, and particularly RET/PTC3, are highly prevalent in radiation induced PTC. However, many thyroid tumors without any history of radiation exposure harbor similar RET rearrangements. The overall prevalence of RET/PTC rearrangements varies from 20 to 70% of PTCs and they are more frequent in childhood than in adulthood thyroid cancer. Controversial data have been reported on the relationship between RET/PTC rearrangements and the PTC prognosis. RET/PTC3 is usually associated with a more aggressive phenotype and in particular with a greater tumor size, the solid variant, and a more advanced stage at diagnosis which are all poor prognostic factors. In contrast, RET/PTC1 rearrangement does not correlate with any clinical–pathological characteristics of PTC. Moreover, the RET protein and mRNA expression level did not show any correlation with the outcome of patients with PTC and no correlation between RET/PTC rearrangements and the

  3. Cell signaling by receptor-tyrosine kinases

    PubMed Central

    Lemmon, Mark A.; Schlessinger, Joseph

    2010-01-01

    Recent structural studies of receptor tyrosine kinases (RTKs) have revealed unexpected diversity in the mechanisms of their activation by growth factor ligands. Strategies for inducing dimerization by ligand binding are surprisingly diverse, as are mechanisms that couple this event to activation of the intracellular tyrosine kinase domains. As our understanding of these details becomes increasingly sophisticated, it provides an important context for therapeutically countering the effects of pathogenic RTK mutations in cancer and other diseases. Much remains to be learned, however, about the complex signaling networks downstream from RTKs and how alterations in these networks are translated into cellular responses. PMID:20602996

  4. Complexity of Receptor Tyrosine Kinase Signal Processing

    PubMed Central

    Volinsky, Natalia; Kholodenko, Boris N.

    2013-01-01

    Our knowledge of molecular mechanisms of receptor tyrosine kinase (RTK) signaling advances with ever-increasing pace. Yet our understanding of how the spatiotemporal dynamics of RTK signaling control specific cellular outcomes has lagged behind. Systems-centered experimental and computational approaches can help reveal how overlapping networks of signal transducers downstream of RTKs orchestrate specific cell-fate decisions. We discuss how RTK network regulatory structures, which involve the immediate posttranslational and delayed transcriptional controls by multiple feed forward and feedback loops together with pathway cross talk, adapt cells to the combinatorial variety of external cues and conditions. This intricate network circuitry endows cells with emerging capabilities for RTK signal processing and decoding. We illustrate how mathematical modeling facilitates our understanding of RTK network behaviors by unraveling specific systems properties, including bistability, oscillations, excitable responses, and generation of intricate landscapes of signaling activities. PMID:23906711

  5. Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase.

    PubMed

    Mithoe, Sharon C; Ludwig, Christina; Pel, Michiel J C; Cucinotta, Mara; Casartelli, Alberto; Mbengue, Malick; Sklenar, Jan; Derbyshire, Paul; Robatzek, Silke; Pieterse, Corné M J; Aebersold, Ruedi; Menke, Frank L H

    2016-03-01

    Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN-SENSITIVE 2 (FLS2) induces the activation of mitogen-activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin-triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7-mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex. PMID:26769563

  6. Identification and characterization of RET fusions in advanced colorectal cancer

    PubMed Central

    Garrett, Christopher R.; Seery, Tara; Sanford, Eric M.; Balasubramanian, Sohail; Ross, Jeffrey S.; Stephens, Philip J.; Miller, Vincent A.; Ali, Siraj M.; Chiu, Vi K.

    2015-01-01

    There is an unmet clinical need for molecularly directed therapies available for metastatic colorectal cancer. Comprehensive genomic profiling has the potential to identify actionable genomic alterations in colorectal cancer. Through comprehensive genomic profiling we prospectively identified 6 RET fusion kinases, including two novel fusions of CCDC6-RET and NCOA4-RET, in metastatic colorectal cancer (CRC) patients. RET fusion kinases represent a novel class of oncogenic driver in CRC and occurred at a 0.2% frequency without concurrent driver mutations, including KRAS, NRAS, BRAF, PIK3CA or other fusion tyrosine kinases. Multiple RET kinase inhibitors were cytotoxic to RET fusion kinase positive cancer cells and not RET fusion kinase negative CRC cells. The presence of a RET fusion kinase may identify a subset of metastatic CRC patients with a high response rate to RET kinase inhibition. This is the first characterization of RET fusions in CRC patients and highlights the therapeutic significance of prospective comprehensive genomic profiling in advanced CRC. PMID:26078337

  7. Identification and characterization of RET fusions in advanced colorectal cancer.

    PubMed

    Le Rolle, Anne-France; Klempner, Samuel J; Garrett, Christopher R; Seery, Tara; Sanford, Eric M; Balasubramanian, Sohail; Ross, Jeffrey S; Stephens, Philip J; Miller, Vincent A; Ali, Siraj M; Chiu, Vi K

    2015-10-01

    There is an unmet clinical need for molecularly directed therapies available for metastatic colorectal cancer. Comprehensive genomic profiling has the potential to identify actionable genomic alterations in colorectal cancer. Through comprehensive genomic profiling we prospectively identified 6 RET fusion kinases, including two novel fusions of CCDC6-RET and NCOA4-RET, in metastatic colorectal cancer (CRC) patients. RET fusion kinases represent a novel class of oncogenic driver in CRC and occurred at a 0.2% frequency without concurrent driver mutations, including KRAS, NRAS, BRAF, PIK3CA or other fusion tyrosine kinases. Multiple RET kinase inhibitors were cytotoxic to RET fusion kinase positive cancer cells and not RET fusion kinase negative CRC cells. The presence of a RET fusion kinase may identify a subset of metastatic CRC patients with a high response rate to RET kinase inhibition. This is the first characterization of RET fusions in CRC patients and highlights the therapeutic significance of prospective comprehensive genomic profiling in advanced CRC. PMID:26078337

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

    PubMed

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

    2015-11-15

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

  9. A Pyrazolo[3,4-d]pyrimidin-4-amine Derivative Containing an Isoxazole Moiety Is a Selective and Potent Inhibitor of RET Gatekeeper Mutants.

    PubMed

    Yoon, Hojong; Kwak, Yeonui; Choi, Seunghye; Cho, Hanna; Kim, Nam Doo; Sim, Taebo

    2016-01-14

    Aberrant RET kinase signaling plays critical roles in several human cancers such as thyroid carcinoma. The gatekeeper mutants (V804L or V804M) of RET are resistant to currently approved RET inhibitors such as cabozantinib and vandetanib. We, for the first time, report a highly selective and extremely potent RET inhibitor, 6i rationally designed. Compound 6i inhibits strongly RET gatekeeper mutants and other clinically relevant RET mutants as well as wt-RET. This substance also significantly suppresses growth of thyroid cancer-derived TT cell lines and Ba/F3 cells transformed with various RET mutants. Docking studies reveal that the isoxazole moiety in 6i is responsible for binding affinity improvement by providing additional site for H-bonding with Lys758. Also, 6i not only substantially blocks cellular RET autophosphorylation and its downstream pathway, it markedly induces apoptosis and anchorage-independent growth inhibition in TT cell lines while having no effect on normal thyroid Nthy ori-3-1 cells. PMID:26652860

  10. Protein kinase A signalling in Schistosoma mansoni cercariae and schistosomules.

    PubMed

    Hirst, Natasha L; Lawton, Scott P; Walker, Anthony J

    2016-06-01

    Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A regulates multiple processes in eukaryotes by phosphorylating diverse cellular substrates, including metabolic and signalling enzymes, ion channels and transcription factors. Here we provide insight into protein kinase A signalling in cercariae and 24h in vitro cultured somules of the blood parasite, Schistosoma mansoni, which causes human intestinal schistosomiasis. Functional mapping of activated protein kinase A using anti-phospho protein kinase A antibodies and confocal laser scanning microscopy revealed activated protein kinase A in the central and peripheral nervous system, oral-tip sensory papillae, oesophagus and excretory system of intact cercariae. Cultured 24h somules, which biologically represent the skin-resident stage of the parasite, exhibited similar activation patterns in oesophageal and nerve tissues but also displayed striking activation at the tegument and activation in a region resembling the germinal 'stem' cell cluster. The adenylyl cyclase activator, forskolin, stimulated somule protein kinase A activation and produced a hyperkinesia phenotype. The biogenic amines, serotonin and dopamine known to be present in skin also induced protein kinase A activation in somules, whereas neuropeptide Y or [Leu(31),Pro(34)]-neuropeptide Y attenuated protein kinase A activation. However, neuropeptide Y did not block the forskolin-induced somule hyperkinesia. Bioinformatic investigation of potential protein associations revealed 193 medium confidence and 59 high confidence protein kinase A interacting partners in S. mansoni, many of which possess putative protein kinase A phosphorylation sites. These data provide valuable insight into the intricacies of protein kinase A signalling in S. mansoni and a framework for further physiological investigations into the roles of protein kinase A in schistosomes, particularly in the context of interactions between the parasite and the host. PMID:26777870

  11. FAK Acts as a Suppressor of RTK-MAP Kinase Signalling in Drosophila melanogaster Epithelia and Human Cancer Cells

    PubMed Central

    Macagno, Juan Pablo; Diaz Vera, Jesica; Yu, Yachuan; MacPherson, Iain; Sandilands, Emma; Palmer, Ruth; Norman, Jim C.; Frame, Margaret; Vidal, Marcos

    2014-01-01

    Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours. PMID:24676055

  12. Feedback Regulation of Kinase Signaling Pathways by AREs and GREs.

    PubMed

    Vlasova-St Louis, Irina; Bohjanen, Paul R

    2016-01-01

    In response to environmental signals, kinases phosphorylate numerous proteins, including RNA-binding proteins such as the AU-rich element (ARE) binding proteins, and the GU-rich element (GRE) binding proteins. Posttranslational modifications of these proteins lead to a significant changes in the abundance of target mRNAs, and affect gene expression during cellular activation, proliferation, and stress responses. In this review, we summarize the effect of phosphorylation on the function of ARE-binding proteins ZFP36 and ELAVL1 and the GRE-binding protein CELF1. The networks of target mRNAs that these proteins bind and regulate include transcripts encoding kinases and kinase signaling pathways (KSP) components. Thus, kinase signaling pathways are involved in feedback regulation, whereby kinases regulate RNA-binding proteins that subsequently regulate mRNA stability of ARE- or GRE-containing transcripts that encode components of KSP. PMID:26821046

  13. Feedback Regulation of Kinase Signaling Pathways by AREs and GREs

    PubMed Central

    Vlasova-St. Louis, Irina; Bohjanen, Paul R.

    2016-01-01

    In response to environmental signals, kinases phosphorylate numerous proteins, including RNA-binding proteins such as the AU-rich element (ARE) binding proteins, and the GU-rich element (GRE) binding proteins. Posttranslational modifications of these proteins lead to a significant changes in the abundance of target mRNAs, and affect gene expression during cellular activation, proliferation, and stress responses. In this review, we summarize the effect of phosphorylation on the function of ARE-binding proteins ZFP36 and ELAVL1 and the GRE-binding protein CELF1. The networks of target mRNAs that these proteins bind and regulate include transcripts encoding kinases and kinase signaling pathways (KSP) components. Thus, kinase signaling pathways are involved in feedback regulation, whereby kinases regulate RNA-binding proteins that subsequently regulate mRNA stability of ARE- or GRE-containing transcripts that encode components of KSP. PMID:26821046

  14. Kinases and kinase signaling pathways: potential therapeutic targets in Parkinson's disease.

    PubMed

    Wang, Gang; Pan, Jing; Chen, Sheng-Di

    2012-08-01

    Complex molecular mechanisms underlying the pathogenesis of Parkinson's disease (PD) are gradually being elucidated. Accumulating genetic evidence implicates dysfunction of kinase activities and phosphorylation pathways in the pathogenesis of PD. Causative and risk gene products associated with PD include protein kinases (such as PINK1, LRRK2 and GAK) and proteins related phosphorylation signaling pathways (such as SNCA, DJ-1). PINK1, LRRK2 and several PD gene products have been associated with mitogen-activated protein (MAP) and protein kinase B (AKT) kinase signaling pathways. C-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERK) and p38, signaling pathways downstream of MAP, are particularly important in PD. JNK and p38 play an integral role in neuronal death. Targeting JNK or p38 signaling may offer an effective therapy for PD. Inhibitors of the ERK signaling pathway, which plays an important role in the development of l-DOPA-induced dyskinesia (LID), have been shown to attenuate this condition in animal models. In this review, we summarize experimental evidence gathered over the last decade on the role of PINK1, LRRK2 and GAK and their related phosphorylation signaling pathways (JNK, ERK, p38 and PI3K/AKT) in PD. It is speculated that improvement or modulation of these signaling pathways will reveal potential therapeutic targets for attenuation of the cardinal symptoms and motor complications in patients with PD in the future. PMID:22709943

  15. 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

  16. 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.

  17. RET fusion gene: translation to personalized lung cancer therapy.

    PubMed

    Kohno, Takashi; Tsuta, Koji; Tsuchihara, Katsuya; Nakaoku, Takashi; Yoh, Kiyotaka; Goto, Koichi

    2013-11-01

    Development of lung adenocarcinoma (LADC), the most frequent histological type of lung cancer, depends in many cases on the activation of "driver" oncogenes such as KRAS, epidermal growth factor receptor (EGFR), and anaplastic lymphoma kinase (ALK). Inhibitors that target the EGFR and ALK tyrosine kinases show therapeutic effects against LADCs containing EGFR gene mutations and ALK gene fusions, respectively. Recently, we and others identified the RET fusion gene as a new targetable driver gene in LADC. The RET fusions occur in 1-2% of LADCs. Existing US Food and Drug Administration-approved inhibitors of RET tyrosine kinase show promising therapeutic effects both in vitro and in vivo, as well as in a few patients. Clinical trials are underway to investigate the therapeutic effects of RET tyrosine kinase inhibitors, such as vandetanib (ZD6474) and cabozantinib (XL184), in patients with RET fusion-positive non-small-cell lung cancer. PMID:23991695

  18. Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling.

    PubMed Central

    Shepherd, P R; Withers, D J; Siddle, K

    1998-01-01

    Insulin plays a key role in regulating a wide range of cellular processes. However, until recently little was known about the signalling pathways that are involved in linking the insulin receptor with downstream responses. It is now apparent that the activation of class 1a phosphoinositide 3-kinase (PI 3-kinase) is necessary and in some cases sufficient to elicit many of insulin's effects on glucose and lipid metabolism. The lipid products of PI 3-kinase act as both membrane anchors and allosteric regulators, serving to localize and activate downstream enzymes and their protein substrates. One of the major ways these lipid products of PI 3-kinase act in insulin signalling is by binding to pleckstrin homology (PH) domains of phosphoinositide-dependent protein kinase (PDK) and protein kinase B (PKB) and in the process regulating the phosphorylation of PKB by PDK. Using mechanisms such as this, PI 3-kinase is able to act as a molecular switch to regulate the activity of serine/threonine-specific kinase cascades important in mediating insulin's effects on endpoint responses. PMID:9677303

  19. Destabilizing RET in targeted treatment of thyroid cancers

    PubMed Central

    Gild, M L; Bullock, M; Pon, C K; Robinson, B G; Clifton-Bligh, R J

    2015-01-01

    Metastatic differentiated thyroid cancers (DTC) are resistant to traditional chemotherapy. Kinase inhibitors have shown promise in patients with progressive DTC, but dose-limiting toxicity is commonplace. HSP90 regulates protein degradation of several growth-mediating kinases such as RET, and we hypothesized that HSP90 inhibitor (AUY922) could inhibit RET-mediated medullary thyroid cancer (MTC) as well as papillary thyroid cancer (PTC) cell growth and also radioactive iodine uptake by PTC cells. Studies utilized MTC cell lines TT (C634W) and MZ-CRC-1 (M918T) and the PTC cell line TPC-1 (RET/PTC1). Cell viability was assessed with MTS assays and apoptosis by flow cytometry. Signaling target expression was determined by western blot and radioiodine uptake measured with a gamma counter. Prolonged treatment of both MTC cell lines with AUY922 simultaneously inhibited both MAPK and mTOR pathways and significantly induced apoptosis (58.7 and 78.7% reduction in MZ-CRC-1 and TT live cells respectively, following 1 μM AUY922; P<0.02). Similarly in the PTC cell line, growth and signaling targets were inhibited, and also a 2.84-fold increase in radioiodine uptake was observed following AUY922 administration (P=0.015). AUY922 demonstrates in vitro activity against MTC and PTC cell lines. We observed a potent dose-dependent increase in apoptosis in MTC cell lines following drug administration confirming its anti-tumorigenic effects. Western blots confirm inhibition of pro-survival proteins including AKT suggesting this as the mechanism of cell death. In a functional study, we observed an increase in radioiodine uptake in the PTC cell line following AUY922 treatment. We believe HSP90 inhibition could be a viable alternative for treatment of RET-driven chemo-resistant thyroid cancers. PMID:26574568

  20. MOLECULAR MECHANISMS OF RECEPTOR KINASE ACTION IN BRASSINOSTEROID SIGNAL TRANSDUCTION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) for hormone perception and signal transduction. To examine early events in BR signaling, we used co-immunoprecipita...

  1. Ret is essential to mediate GDNF's neuroprotective and neuroregenerative effect in a Parkinson disease mouse model.

    PubMed

    Drinkut, Anja; Tillack, Karsten; Meka, Durga P; Schulz, Jorg B; Kügler, Sebastian; Kramer, Edgar R

    2016-01-01

    Glial cell line-derived neurotrophic factor (GDNF) is a potent survival and regeneration-promoting factor for dopaminergic neurons in cell and animal models of Parkinson disease (PD). GDNF is currently tested in clinical trials on PD patients with so far inconclusive results. The receptor tyrosine kinase Ret is the canonical GDNF receptor, but several alternative GDNF receptors have been proposed, raising the question of which signaling receptor mediates here the beneficial GDNF effects. To address this question we overexpressed GDNF in the striatum of mice deficient for Ret in dopaminergic neurons and subsequently challenged these mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Strikingly, in this established PD mouse model, the absence of Ret completely abolished GDNF's neuroprotective and regenerative effect on the midbrain dopaminergic system. This establishes Ret signaling as absolutely required for GDNF's effects to prevent and compensate dopaminergic system degeneration and suggests Ret activation as the primary target of GDNF therapy in PD. PMID:27607574

  2. Ret and Etv4 Promote Directed Movements of Progenitor Cells during Renal Branching Morphogenesis.

    PubMed

    Riccio, Paul; Cebrian, Cristina; Zong, Hui; Hippenmeyer, Simon; Costantini, Frank

    2016-02-01

    Branching morphogenesis of the epithelial ureteric bud forms the renal collecting duct system and is critical for normal nephron number, while low nephron number is implicated in hypertension and renal disease. Ureteric bud growth and branching requires GDNF signaling from the surrounding mesenchyme to cells at the ureteric bud tips, via the Ret receptor tyrosine kinase and coreceptor Gfrα1; Ret signaling up-regulates transcription factors Etv4 and Etv5, which are also critical for branching. Despite extensive knowledge of the genetic control of these events, it is not understood, at the cellular level, how renal branching morphogenesis is achieved or how Ret signaling influences epithelial cell behaviors to promote this process. Analysis of chimeric embryos previously suggested a role for Ret signaling in promoting cell rearrangements in the nephric duct, but this method was unsuited to study individual cell behaviors during ureteric bud branching. Here, we use Mosaic Analysis with Double Markers (MADM), combined with organ culture and time-lapse imaging, to trace the movements and divisions of individual ureteric bud tip cells. We first examine wild-type clones and then Ret or Etv4 mutant/wild-type clones in which the mutant and wild-type sister cells are differentially and heritably marked by green and red fluorescent proteins. We find that, in normal kidneys, most individual tip cells behave as self-renewing progenitors, some of whose progeny remain at the tips while others populate the growing UB trunks. In Ret or Etv4 MADM clones, the wild-type cells generated at a UB tip are much more likely to remain at, or move to, the new tips during branching and elongation, while their Ret-/- or Etv4-/- sister cells tend to lag behind and contribute only to the trunks. By tracking successive mitoses in a cell lineage, we find that Ret signaling has little effect on proliferation, in contrast to its effects on cell movement. Our results show that Ret/Etv4 signaling

  3. Signal Transduction in Histidine Kinases: Insights from New Structures

    PubMed Central

    Bhate, Manasi P.; Molnar, Kathleen S.; Goulian, Mark; DeGrado, William F.

    2015-01-01

    Histidine kinases (HKs) are major players in bacterial signaling. There has been an explosion of new HK crystal structures in the last five years. We globally analyze the structures of HKs to yield insights into the mechanisms by which signals are transmitted to and across protein structures in this family. We interpret known enzymological data in the context of new structural data to show how asymmetry across the dimer interface is a key feature of signal transduction in HKs, and discuss how different HK domains undergo asymmetric-to-symmetric transitions during signal transduction and catalysis. A thermodynamic framework for signaling that encompasses these various properties is presented and the consequences of weak thermodynamic coupling are discussed. The synthesis of observations from enzymology, structural biology, protein engineering and thermodynamics paves the way for a deeper molecular understanding of histidine kinase signal transduction. PMID:25982528

  4. Adenylate Kinase and AMP Signaling Networks: Metabolic Monitoring, Signal Communication and Body Energy Sensing

    PubMed Central

    Dzeja, Petras; Terzic, Andre

    2009-01-01

    Adenylate kinase and downstream AMP signaling is an integrated metabolic monitoring system which reads the cellular energy state in order to tune and report signals to metabolic sensors. A network of adenylate kinase isoforms (AK1-AK7) are distributed throughout intracellular compartments, interstitial space and body fluids to regulate energetic and metabolic signaling circuits, securing efficient cell energy economy, signal communication and stress response. The dynamics of adenylate kinase-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, cell and ciliary motility, nuclear transport, energetics of cell cycle, DNA synthesis and repair, and developmental programming. Metabolomic analyses indicate that cellular, interstitial and blood AMP levels are potential metabolic signals associated with vital functions including body energy sensing, sleep, hibernation and food intake. Either low or excess AMP signaling has been linked to human disease such as diabetes, obesity and hypertrophic cardiomyopathy. Recent studies indicate that derangements in adenylate kinase-mediated energetic signaling due to mutations in AK1, AK2 or AK7 isoforms are associated with hemolytic anemia, reticular dysgenesis and ciliary dyskinesia. Moreover, hormonal, food and antidiabetic drug actions are frequently coupled to alterations of cellular AMP levels and associated signaling. Thus, by monitoring energy state and generating and distributing AMP metabolic signals adenylate kinase represents a unique hub within the cellular homeostatic network. PMID:19468337

  5. Phosphatidylinositol 3-kinase signaling determines kidney size

    PubMed Central

    Chen, Jian-Kang; Nagai, Kojiro; Chen, Jianchun; Plieth, David; Hino, Masayo; Xu, Jinxian; Sha, Feng; Ikizler, T. Alp; Quarles, C. Chad; Threadgill, David W.; Neilson, Eric G.; Harris, Raymond C.

    2015-01-01

    Kidney size adaptively increases as mammals grow and in response to the loss of 1 kidney. It is not clear how kidneys size themselves or if the processes that adapt kidney mass to lean body mass also mediate renal hypertrophy following unilateral nephrectomy (UNX). Here, we demonstrated that mice harboring a proximal tubule–specific deletion of Pten (PtenptKO) have greatly enlarged kidneys as the result of persistent activation of the class I PI3K/mTORC2/AKT pathway and an increase of the antiproliferative signals p21Cip1/WAF and p27Kip1. Administration of rapamycin to PtenptKO mice diminished hypertrophy. Proximal tubule–specific deletion of Egfr in PtenptKO mice also attenuated class I PI3K/mTORC2/AKT signaling and reduced the size of enlarged kidneys. In PtenptKO mice, UNX further increased mTORC1 activation and hypertrophy in the remaining kidney; however, mTORC2-dependent AKT phosphorylation did not increase further in the remaining kidney of PtenptKO mice, nor was it induced in the remaining kidney of WT mice. After UNX, renal blood flow and amino acid delivery to the remaining kidney rose abruptly, followed by increased amino acid content and activation of a class III PI3K/mTORC1/S6K1 pathway. Thus, our findings demonstrate context-dependent roles for EGFR-modulated class I PI3K/mTORC2/AKT signaling in the normal adaptation of kidney size and PTEN-independent, nutrient-dependent class III PI3K/mTORC1/S6K1 signaling in the compensatory enlargement of the remaining kidney following UNX. PMID:25985273

  6. Ret and Etv4 Promote Directed Movements of Progenitor Cells during Renal Branching Morphogenesis

    PubMed Central

    Riccio, Paul; Cebrian, Cristina; Zong, Hui; Hippenmeyer, Simon; Costantini, Frank

    2016-01-01

    Branching morphogenesis of the epithelial ureteric bud forms the renal collecting duct system and is critical for normal nephron number, while low nephron number is implicated in hypertension and renal disease. Ureteric bud growth and branching requires GDNF signaling from the surrounding mesenchyme to cells at the ureteric bud tips, via the Ret receptor tyrosine kinase and coreceptor Gfrα1; Ret signaling up-regulates transcription factors Etv4 and Etv5, which are also critical for branching. Despite extensive knowledge of the genetic control of these events, it is not understood, at the cellular level, how renal branching morphogenesis is achieved or how Ret signaling influences epithelial cell behaviors to promote this process. Analysis of chimeric embryos previously suggested a role for Ret signaling in promoting cell rearrangements in the nephric duct, but this method was unsuited to study individual cell behaviors during ureteric bud branching. Here, we use Mosaic Analysis with Double Markers (MADM), combined with organ culture and time-lapse imaging, to trace the movements and divisions of individual ureteric bud tip cells. We first examine wild-type clones and then Ret or Etv4 mutant/wild-type clones in which the mutant and wild-type sister cells are differentially and heritably marked by green and red fluorescent proteins. We find that, in normal kidneys, most individual tip cells behave as self-renewing progenitors, some of whose progeny remain at the tips while others populate the growing UB trunks. In Ret or Etv4 MADM clones, the wild-type cells generated at a UB tip are much more likely to remain at, or move to, the new tips during branching and elongation, while their Ret−/− or Etv4−/− sister cells tend to lag behind and contribute only to the trunks. By tracking successive mitoses in a cell lineage, we find that Ret signaling has little effect on proliferation, in contrast to its effects on cell movement. Our results show that Ret/Etv4

  7. RET mutations in MEN 2 associated diseases

    SciTech Connect

    Hofstra, R.M.W.; Stelwagen, T.; Stulp, R.P.

    1994-09-01

    Multiple endocrine neoplasia type 2 (MEN 2) comprises three clinically distinct dominantly inherited cancer syndromes namely MEN 2A, MEN 2B and familial medullary thyroid carcinoma (FMTC). Germline (point) mutations of the RET proto-oncogene have been reported to occur in all these syndromes. In MEN 2A and FMTC patients the mutations occurred within codons specifying cysteine residues in the transition of the RET extracellular and transmembrane domains, while in MEN 2B patients we could detect a single RET mutation in the tyrosine kinase domain in all patients. Also in patients suffering from Hirschsprung`s disease (HSCR), mutations in the RET gene have been found. These mutations are spread all over the gene. Several families have been described in which MEN 2 and HSCR are associated. MEN 2A is also found associated with cutaneous lichen amyloidosis (CLA). It might be that specific RET mutations correlate with these disease associations. We therefore scanned DNA from patients from a family with MEN 2A and HSCR, MEN 2A and CLA and CLA only for RET mutations. Results obtained thus far do not support the existence of specific correlations.

  8. Genetic variation in insulin-induced kinase signaling

    PubMed Central

    Wang, Isabel Xiaorong; Ramrattan, Girish; Cheung, Vivian G

    2015-01-01

    Individual differences in sensitivity to insulin contribute to disease susceptibility including diabetes and metabolic syndrome. Cellular responses to insulin are well studied. However, which steps in these response pathways differ across individuals remains largely unknown. Such knowledge is needed to guide more precise therapeutic interventions. Here, we studied insulin response and found extensive individual variation in the activation of key signaling factors, including ERK whose induction differs by more than 20-fold among our subjects. This variation in kinase activity is propagated to differences in downstream gene expression response to insulin. By genetic analysis, we identified cis-acting DNA variants that influence signaling response, which in turn affects downstream changes in gene expression and cellular phenotypes, such as protein translation and cell proliferation. These findings show that polymorphic differences in signal transduction contribute to individual variation in insulin response, and suggest kinase modulators as promising therapeutics for diseases characterized by insulin resistance. PMID:26202599

  9. Novel links in the plant TOR kinase signaling network.

    PubMed

    Xiong, Yan; Sheen, Jen

    2015-12-01

    Nutrient and energy sensing and signaling mechanisms constitute the most ancient and fundamental regulatory networks to control growth and development in all life forms. The target of rapamycin (TOR) protein kinase is modulated by diverse nutrient, energy, hormone and stress inputs and plays a central role in regulating cell proliferation, growth, metabolism and stress responses from yeasts to plants and animals. Recent chemical, genetic, genomic and metabolomic analyses have enabled significant progress toward molecular understanding of the TOR signaling network in multicellular plants. This review discusses the applications of new chemical tools to probe plant TOR functions and highlights recent findings and predictions on TOR-mediate biological processes. Special focus is placed on novel and evolutionarily conserved TOR kinase effectors as positive and negative signaling regulators that control transcription, translation and metabolism to support cell proliferation, growth and maintenance from embryogenesis to senescence in the plant system. PMID:26476687

  10. Information transfer by leaky, heterogeneous, protein kinase signaling systems

    PubMed Central

    Voliotis, Margaritis; Perrett, Rebecca M.; McWilliams, Chris; McArdle, Craig A.; Bowsher, Clive G.

    2014-01-01

    Cells must sense extracellular signals and transfer the information contained about their environment reliably to make appropriate decisions. To perform these tasks, cells use signal transduction networks that are subject to various sources of noise. Here, we study the effects on information transfer of two particular types of noise: basal (leaky) network activity and cell-to-cell variability in the componentry of the network. Basal activity is the propensity for activation of the network output in the absence of the signal of interest. We show, using theoretical models of protein kinase signaling, that the combined effect of the two types of noise makes information transfer by such networks highly vulnerable to the loss of negative feedback. In an experimental study of ERK signaling by single cells with heterogeneous ERK expression levels, we verify our theoretical prediction: In the presence of basal network activity, negative feedback substantially increases information transfer to the nucleus by both preventing a near-flat average response curve and reducing sensitivity to variation in substrate expression levels. The interplay between basal network activity, heterogeneity in network componentry, and feedback is thus critical for the effectiveness of protein kinase signaling. Basal activity is widespread in signaling systems under physiological conditions, has phenotypic consequences, and is often raised in disease. Our results reveal an important role for negative feedback mechanisms in protecting the information transfer function of saturable, heterogeneous cell signaling systems from basal activity. PMID:24395805

  11. MELK-a conserved kinase: functions, signaling, cancer, and controversy.

    PubMed

    Ganguly, Ranjit; Mohyeldin, Ahmed; Thiel, Jordyn; Kornblum, Harley I; Beullens, Monique; Nakano, Ichiro

    2015-01-01

    Maternal embryonic leucine zipper kinase (MELK) is a highly conserved serine/threonine kinase initially found to be expressed in a wide range of early embryonic cellular stages, and as a result has been implicated in embryogenesis and cell cycle control. Recent evidence has identified a broader spectrum of tissue expression pattern for this kinase than previously appreciated. MELK is expressed in several human cancers and stem cell populations. Unique spatial and temporal patterns of expression within these tissues suggest that MELK plays a prominent role in cell cycle control, cell proliferation, apoptosis, cell migration, cell renewal, embryogenesis, oncogenesis, and cancer treatment resistance and recurrence. These findings have important implications for our understanding of development, disease, and cancer therapeutics. Furthermore understanding MELK signaling may elucidate an added dimension of stem cell control. PMID:25852826

  12. Anaplastic Lymphoma Kinase (ALK) Signaling in Lung Cancer.

    PubMed

    Ou, Sai-Hong Ignatius; Shirai, Keisuke

    2016-01-01

    Chromosomal rearrangement in the anaplastic lymphoma kinase (ALK) gene was identified as an oncogenic driver in non-small cell lung cancer (NSCLC) in 2007. A multi-targeted ALK/ROS1/MET inhibitor, crizotinib, targeting this activated tyrosine kinase has led to significant clinical benefit including tumor shrinkage and prolonged survival without disease progression and has been approved by US FDA since 2011 for the treatment of advanced ALK-rearranged NSCLC (Ou et al. Oncologist 17:1351-1375, 2012). Knowledge gained from treating ALK-rearranged NSCLC patients including the presenting clinicopathologic characteristics, methods of detecting ALK-rearranged NSCLC, pattern of relapse and acquired resistance mechanisms while on crizotinib, and the clinical activities of more potent ALK inhibitors has led us to a detailed and ever expanding knowledge of the ALK signaling pathway in lung cancer but also raising many more questions that remained to be answered in the future. This book chapter will provide a concise summary of the importance of ALK signaling pathway in lung cancer. Understanding the ALK signaling pathway in lung cancer will likely provide the roadmap to the management of major epithelial malignancies driven by receptor tyrosine kinase rearrangement. PMID:26667344

  13. Protein kinase A signaling during bidirectional axenic differentiation in Leishmania.

    PubMed

    Bachmaier, Sabine; Witztum, Ronit; Tsigankov, Polina; Koren, Roni; Boshart, Michael; Zilberstein, Dan

    2016-02-01

    Parasitic protozoa of the genus Leishmania are obligatory intracellular parasites that cycle between the phagolysosome of mammalian macrophages, where they proliferate as intracellular amastigotes, and the midgut of female sand flies, where they proliferate as extracellular promastigotes. Shifting between the two environments induces signaling pathway-mediated developmental processes that enable adaptation to both host and vector. Developmentally regulated expression and phosphorylation of protein kinase A subunits in Leishmania and in Trypanosoma brucei point to an involvement of protein kinase A in parasite development. To assess this hypothesis in Leishmania donovani, we determined proteome-wide changes in phosphorylation of the conserved protein kinase A phosphorylation motifs RXXS and RXXT, using a phospho-specific antibody. Rapid dephosphorylation of these motifs was observed upon initiation of promastigote to amastigote differentiation in culture. No phosphorylated sites were detected in axenic amastigotes. To analyse the kinetics of (re)phosphorylation during axenic reverse differentiation from L. donovani amastigotes to promastigotes, we first established a map of this process with morphological and molecular markers. Upon initiation, the parasites rested for 6-12h before proliferation of an asynchronous population resumed. After early changes in cell shape, the major changes in molecular marker expression and flagella biogenesis occurred between 24 and 33h after initiation. RXXS/T re-phosphorylation and expression of the regulatory subunit PKAR1 correlated with promastigote maturation, indicating a promastigote-specific function of protein kinase A signaling. This is supported by the localization of PKAR1 to the flagellum, an organelle reduced to a remnant in amastigote forms. We conclude that a significant increase in protein kinase A-mediated phosphorylation is part of the ordered changes that characterise the amastigote to promastigote differentiation

  14. Receptor tyrosine kinases: mechanisms of activation and signaling

    PubMed Central

    Hubbard, Stevan R.; Miller, W. Todd

    2008-01-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication. These single-pass transmembrane receptors, which bind polypeptide ligands — mainly growth factors — play key roles in processes such as cellular growth, differentiation, metabolism and motility. Recent progress has been achieved towards an understanding of the precise (and varied) mechanisms by which RTKs are activated by ligand binding and by which signals are propagated from the activated receptors to downstream targets in the cell. PMID:17306972

  15. Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways.

    PubMed

    Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P; Taub, Dennis D

    2014-12-20

    Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levels and impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

  16. Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

    PubMed Central

    Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

    2014-01-01

    Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

  17. Some implications of receptor kinase signaling pathway for development of multitargeted kinase inhibitors.

    PubMed

    Mitrasinovic, Petar M

    2013-03-01

    Epidermal growth factor receptors (EGFRs) belong to the ErbB family of receptor tyrosine kinases (TKs). Based on the role of EGFR signaling pathway in malignant progression of various types of tumors, a growing interest in the use of EGFR-TK inhibitors as probes for molecular imaging of EGFR-overexpressing tumors via positron emission tomography (PET) and single photon emission computed tomography (SPECT) is being notable. On one side, such noninvasive and repetitive monitoring of the activity of EGFR at the kinase level is intended to provide a direct measure of EGFR occupancy and inhibition by EGFR-targeting drugs. On the other side, all oncologic imaging tracers are molecularly targeted radiopharmaceuticals, which are strongly dependent on the tumor biochemistry including increased metabolism, hyperproliferation, angiogenesis, hypoxia, apoptosis, and specific tumor biomarkers (tumor specific antigens and tumor-specific receptors). The present article is an attempt to reconcile these two vital standpoints influencing the choice of appropriate radiolabeled agents for PET and SPECT imaging aimed to support the development of a new generation of multi-targeted kinase inhibitors in the time ahead, because the routine accomplishment of drug selectivity for particular protein kinases is a substantial challenge. PMID:23278847

  18. Cell signaling through protein kinase C oxidation and activation.

    PubMed

    Cosentino-Gomes, Daniela; Rocco-Machado, Nathália; Meyer-Fernandes, José Roberto

    2012-01-01

    Due to the growing importance of cellular signaling mediated by reactive oxygen species (ROS), proteins that are reversibly modulated by these reactant molecules are of high interest. In this context, protein kinases and phosphatases, which act coordinately in the regulation of signal transduction through the phosphorylation and dephosphorylation of target proteins, have been described to be key elements in ROS-mediated signaling events. The major mechanism by which these proteins may be modified by oxidation involves the presence of key redox-sensitive cysteine residues. Protein kinase C (PKC) is involved in a variety of cellular signaling pathways. These proteins have been shown to contain a unique structural feature that is susceptible to oxidative modification. A large number of scientific studies have highlighted the importance of ROS as a second messenger in numerous cellular processes, including cell proliferation, gene expression, adhesion, differentiation, senescence, and apoptosis. In this context, the goal of this review is to discuss the mechanisms by which PKCs are modulated by ROS and how these processes are involved in the cellular response. PMID:23109817

  19. Ribosomal protein S6 kinase 1 signaling regulates mammalian lifespan

    PubMed Central

    Selman, Colin; Tullet, Jennifer M.A.; Wieser, Daniela; Irvine, Elaine; Lingard, Steven J.; Choudhury, Agharul I.; Claret, Marc; Al-Qassab, Hind; Carmignac, Danielle; Ramadani, Faruk; Woods, Angela; Robinson, Iain C.A.; Schuster, Eugene; Batterham, Rachel L.; Kozma, Sara C.; Thomas, George; Carling, David; Okkenhaug, Klaus; Thornton, Janet M.; Partridge, Linda; Gems, David; Withers, Dominic J.

    2016-01-01

    Caloric restriction (CR) protects against aging and disease but the mechanisms by which this affects mammalian lifespan are unclear. We show in mice that deletion of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway component ribosomal S6 protein kinase 1 (S6K1) led to increased lifespan and resistance to age-related pathologies such as bone, immune and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian lifespan, and suggest therapeutic manipulation of S6K1 and AMPK might mimic CR and provide broad protection against diseases of aging. PMID:19797661

  20. 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

  1. Intramolecular conformational changes optimize protein kinase C signaling.

    PubMed

    Antal, Corina E; Violin, Jonathan D; Kunkel, Maya T; Skovsø, Søs; Newton, Alexandra C

    2014-04-24

    Optimal tuning of enzyme signaling is critical for cellular homeostasis. We use fluorescence resonance energy transfer reporters in live cells to follow conformational transitions that tune the affinity of a multidomain signal transducer, protein kinase C (PKC), for optimal response to second messengers. This enzyme comprises two diacylglycerol sensors, the C1A and C1B domains, that have a sufficiently high intrinsic affinity for ligand so that the enzyme would be in a ligand-engaged, active state if not for mechanisms that mask its domains. We show that both diacylglycerol sensors are exposed in newly synthesized PKC and that conformational transitions following priming phosphorylations mask the domains so that the lower affinity sensor, the C1B domain, is the primary diacylglycerol binder. The conformational rearrangements of PKC serve as a paradigm for how multimodule transducers optimize their dynamic range of signaling. PMID:24631122

  2. Oscillatory Dynamics of the Extracellular Signal-regulated Kinase Pathway

    SciTech Connect

    Shankaran, Harish; Wiley, H. S.

    2010-12-01

    The extracellular signal-regulated kinase (ERK) pathway is a central signaling pathway in development and disease and is regulated by multiple negative and positive feedback loops. Recent studies have shown negative feedback from ERK to upstream regulators can give rise to biochemical oscillations with a periodicity of between 15-30 minutes. Feedback due to the stimulated transcription of negative regulators of the ERK pathway can also give rise to transcriptional oscillations with a periodicity of 1-2h. The biological significance of these oscillations is not clear, but recent evidence suggests that transcriptional oscillations participate in developmental processes, such as somite formation. Biochemical oscillations are more enigmatic, but could provide a mechanism for encoding different types of inputs into a common signaling pathway.

  3. Antigen receptor signaling: integration of protein tyrosine kinase functions.

    PubMed

    Tamir, I; Cambier, J C

    1998-09-17

    Antigen receptors on T and B cells function to transduce signals leading to a variety of biologic responses minimally including antigen receptor editing, apoptotic death, developmental progression, cell activation, proliferation and survival. The response to antigen depends upon antigen affinity and valence, involvement of coreceptors in signaling and differentiative stage of the responding cell. The requirement that these receptors integrate signals that drive an array of responses may explain their evolved structural complexity. Antigen receptors are composed of multiple subunits compartmentalized to provide antigen recognition and signal transduction function. In lieu of on-board enzymatic activity these receptors rely on associated Protein Tyrosine Kinases (PTKs) for their signaling function. By aggregating the receptors, and hence their appended PTKs, antigens induce PTK transphosphorylation, activating them to phosphorylate the receptor within conserved motifs termed Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) found in transducer subunits. The tyrosyl phosphorylated ITAMs then interact with Src Homology 2 (SH2) domains within the PTKs leading to their further activation. As receptor phosphorylation is amplified, other effectors, such as Shc, dock by virtue of SH2 binding, and serve, in-turn, as substrates for these PTKs. This sequence of events not only provides a signal amplification mechanism by combining multiple consecutive steps with positive feedback, but also allows for signal diversification by differential recruitment of effectors that provide access to distinct parallel downstream signaling pathways. The subject of antigen receptor signaling has been recently reviewed in depth (DeFranco, 1997; Kurosaki, 1997). Here we discuss the biochemical basis of antigen receptor signal transduction, using the B cell receptor (BCR) as a paradigm, with specific emphasis on the involved PTKs. We review several specific mechanisms by which responses

  4. Mitogen-activated protein kinase kinase kinase 1 (MAP3K1) integrates developmental signals for eyelid closure

    PubMed Central

    Geh, Esmond; Meng, Qinghang; Mongan, Maureen; Wang, Jingcai; Takatori, Atsushi; Zheng, Yi; Puga, Alvaro; Lang, Richard A.; Xia, Ying

    2011-01-01

    Developmental eyelid closure is an evolutionarily conserved morphogenetic event requiring proliferation, differentiation, cytoskeleton reorganization, and migration of epithelial cells at the tip of the developing eyelid. Many signaling events take place during eyelid closure, but how the signals converge to regulate the morphogenetic process remains an open and intriguing question. Here we show that mitogen-activated protein kinase kinase kinase 1 (MAP3K1) highly expressed in the developing eyelid epithelium, forms with c-Jun, a regulatory axis that orchestrates morphogenesis by integrating two different networks of eyelid closure signals. A TGF-α/EGFR-RhoA module initiates one of these networks by inducing c-Jun expression which, in a phosphorylation-independent manner, binds to the Map3k1 promoter and causes an increase in MAP3K1 expression. RhoA knockout in the ocular surface epithelium disturbs this network by decreasing MAP3K1 expression, and causes delayed eyelid closure in Map3k1 hemizygotes. The second network is initiated by the enzymatic activity of MAP3K1, which phosphorylates and activates a JNK-c-Jun module, leading to AP-1 transactivation and induction of its downstream genes, such as Pai-1. MAP3K1 inactivation reduces AP-1 activity and PAI-1 expression both in cells and developing eyelids. MAP3K1 is therefore the nexus of an intracrine regulatory loop connecting the TGF-α/EGFR/RhoA-c-Jun and JNK-c-Jun-AP-1 pathways in developmental eyelid closure. PMID:21969564

  5. GSK-3 kinases enhance calcineurin signaling by phosphorylation of RCNs

    PubMed Central

    Hilioti, Zoe; Gallagher, Deirdre A.; Low-Nam, Shalini T.; Ramaswamy, Priya; Gajer, Pawel; Kingsbury, Tami J.; Birchwood, Christine J.; Levchenko, Andre; Cunningham, Kyle W.

    2004-01-01

    The conserved RCN family of proteins can bind and directly regulate calcineurin, a Ca2+-activated protein phosphatase involved in immunity, heart growth, muscle development, learning, and other processes. Whereas high levels of RCNs can inhibit calcineurin signaling in fungal and animal cells, RCNs can also stimulate calcineurin signaling when expressed at endogenous levels. Here we show that the stimulatory effect of yeast Rcn1 involves phosphorylation of a conservedserine residue by Mck1, a member of the GSK-3 family of protein kinases. Mutations at the GSK-3 consensus site of Rcn1 and human DSCR1/MCIP1 abolish the stimulatory effects on calcineurin signaling. RCNs may therefore oscillate between stimulatory and inhibitory forms in vivo in a manner similar to the Inhibitor-2 regulators of type 1 protein phosphatase. Computational modeling indicates a biphasic response of calcineurin to increasing RCN concentration such that protein phosphatase activity is stimulated by low concentrations of phospho-RCN and inhibited by high concentrations of phospho- or dephospho-RCN. This prediction was verified experimentally in yeast cells expressing Rcn1 or DSCR1/MCIP1 at different concentrations. Through the phosphorylation of RCNs, GSK-3 kinases can potentially contribute to a positive feedback loop involving calcineurin-dependent up-regulation of RCN expression. Such feedback may help explain the large induction of DSCR1/MCIP1 observed in brain of Down syndrome individuals. PMID:14701880

  6. Signals fly when kinases meet Rho-of-plants (ROP) small G-proteins.

    PubMed

    Fehér, Attila; Lajkó, Dézi Bianka

    2015-08-01

    Rho-type small GTP-binding plant proteins function as two-state molecular switches in cellular signalling. There is accumulating evidence that Rho-of-plants (ROP) signalling is positively controlled by plant receptor kinases, through the ROP guanine nucleotide exchange factor proteins. These signalling modules regulate cell polarity, cell shape, hormone responses, and pathogen defence, among other things. Other ROP-regulatory proteins might also be subjected to protein phosphorylation by cellular kinases (e.g., mitogen-activated protein kinases or calcium-dependent protein kinases), in order to integrate various cellular signalling pathways with ROP GTPase-dependent processes. In contrast to the role of kinases in upstream ROP regulation, much less is known about the potential link between ROP GTPases and downstream kinase signalling. In other eukaryotes, Rho-type G-protein-activated kinases are widespread and have a key role in many cellular processes. Recent data indicate the existence of structurally different ROP-activated kinases in plants, but their ROP-dependent biological functions still need to be validated. In addition to these direct interactions, ROPs may also indirectly control the activity of mitogen-activated protein kinases or calcium-dependent protein kinases. These kinases may therefore function as upstream as well as downstream kinases in ROP-mediated signalling pathways, such as the phosphatidylinositol monophosphate kinases involved in cell polarity establishment. PMID:26089155

  7. RET-targeting molecular stratified non-small-cell lung cancers

    PubMed Central

    2013-01-01

    Recent advances in lung cancer genomics have successfully characterized therapeutic targets of lung cancer. RET fusion gene products are among the newest target molecules for lung adenocarcinoma. Preclinical findings and preliminary reports regarding potential tumor control by RET-targeting multi-kinase inhibitors encourage further clinical trials. The infrequent prevalence of RET fusion gene-positive cases may be a major obstacle hindering the development of RET-targeted therapy. Thus, it is necessary to recruit appropriate participants for trials to develop an efficient RET fusion gene detection system to achieve targeted therapy for lung adenocarcinomas stratified by this molecular target. PMID:25806272

  8. Diversity of mutations in the RET proto-oncogene and its oncogenic mechanism in medullary thyroid cancer.

    PubMed

    Hedayati, Mehdi; Zarif Yeganeh, Marjan; Sheikholeslami, Sara; Afsari, Farinaz

    2016-08-01

    Thyroid cancer is the most common endocrine malignancy and accounts for nearly 1% of all of human cancer. Thyroid cancer has four main histological types: papillary, follicular, medullary, and anaplastic. Papillary, follicular, and anaplastic thyroid carcinomas are derived from follicular thyroid cells, whereas medullary thyroid carcinoma (MTC) originates from the neural crest parafollicular cells or C-cells of the thyroid gland. MTC represents a neuroendocrine tumor and differs considerably from differentiated thyroid carcinoma. MTC is one of the aggressive types of thyroid cancer, which represents 3-10% of all thyroid cancers. It occurs in hereditary (25%) and sporadic (75%) forms. The hereditary form of MTC has an autosomal dominant mode of inheritance. According to the present classification, hereditary MTC is classified as a multiple endocrine neoplasi type 2 A & B (MEN2A & MEN2B) and familial MTC (FMTC). The RET proto-oncogene is located on chromosome 10q11.21. It is composed of 21 exons and encodes a transmembrane receptor tyrosine kinase. RET regulates a complex network of signal transduction pathways during development, survival, proliferation, differentiation, and migration of the enteric nervous system progenitor cells. Gain of function mutations in RET have been well demonstrated in MTC development. Variants of MTC result from different RET mutations, and they have a good genotype-phenotype correlation. Various MTC related mutations have been reported in different exons of the RET gene. We proposed that RET genetic mutations may be different in distinct populations. Therefore, the aim of this study was to find a geographical pattern of RET mutations in different populations. PMID:26678667

  9. RET/PTC rearrangements preferentially occurred in papillary thyroid cancer among atomic bomb survivors exposed to high radiation dose.

    PubMed

    Hamatani, Kiyohiro; Eguchi, Hidetaka; Ito, Reiko; Mukai, Mayumi; Takahashi, Keiko; Taga, Masataka; Imai, Kazue; Cologne, John; Soda, Midori; Arihiro, Koji; Fujihara, Megumu; Abe, Kuniko; Hayashi, Tomayoshi; Nakashima, Masahiro; Sekine, Ichiro; Yasui, Wataru; Hayashi, Yuzo; Nakachi, Kei

    2008-09-01

    A major early event in papillary thyroid carcinogenesis is constitutive activation of the mitogen-activated protein kinase signaling pathway caused by alterations of a single gene, typically rearrangements of the RET and NTRK1 genes or point mutations in the BRAF and RAS genes. In childhood papillary thyroid cancer, regardless of history of radiation exposure, RET/PTC rearrangements are a major event. Conversely, in adult-onset papillary thyroid cancer among the general population, the most common molecular event is BRAF(V600E) point mutation, not RET/PTC rearrangements. To clarify which gene alteration, chromosome aberration, or point mutation preferentially occurs in radiation-associated adult-onset papillary thyroid cancer, we have performed molecular analyses on RET/PTC rearrangements and BRAF(V600E) mutation in 71 papillary thyroid cancer cases among atomic bomb survivors (including 21 cases not exposed to atomic bomb radiation), in relation to radiation dose as well as time elapsed since atomic bomb radiation exposure. RET/PTC rearrangements showed significantly increased frequency with increased radiation dose (P(trend) = 0.002). In contrast, BRAF(V600E) mutation was less frequent in cases exposed to higher radiation dose (P(trend) < 0.001). Papillary thyroid cancer subjects harboring RET/PTC rearrangements developed this cancer earlier than did cases with BRAF(V600E) mutation (P = 0.03). These findings were confirmed by multivariate logistic regression analysis. These results suggest that RET/PTC rearrangements play an important role in radiation-associated thyroid carcinogenesis. PMID:18757433

  10. RET-rearranged non-small-cell lung carcinoma: a clinicopathological and molecular analysis

    PubMed Central

    Tsuta, K; Kohno, T; Yoshida, A; Shimada, Y; Asamura, H; Furuta, K; Kushima, R

    2014-01-01

    Background: To elucidate clinicopathological characteristics of non-small-cell lung carcinoma (NSCLC) cases carrying RET rearrangements causing oncogenic fusions to identify responders to therapy with RET tyrosine kinase inhibitors. Methods: We investigated 1874 patients with carcinomas, including 1620 adenocarcinomas (ADCs), 203 squamous cell carcinomas (SCCs), 8 large cell carcinomas, and 43 sarcomatoid carcinomas (SACs). Fluorescence in situ hybridisation (FISH) and/or reverse transcription–PCR (RT–PCR) were performed to detect RET gene rearrangement. Results: In all, 22 cases (1.2%) showed RET rearrangements; all cases were of ADC histology. Of the 22 patients, 19 possessed KIF5B–RET fusion genes, whereas 3 possessed CCDC6–RET fusion genes. The RET-rearranged tumours were significantly more common in younger patients (P=0.038) and tended to occur in patients with no history of smoking (P=0.051). In addition, RET rearrangements were not associated with gender, occupational history (particularly radioactive exposure), tumour size, lymph node status, tumour stage, or patient survival. The predominant growth pattern in RET-rearranged ADCs was lepidic in 6 cases, papillary in 9 cases, acinar in 2 cases, micropapillary in 1 case, and solid in 4 cases. Cells with cytoplasmic mucin production were at least focally present in 12 of the 22 (54.5%) RET-rearranged ADC cases. Among the 21 analysed RET-rearranged tumours, RET immunopositivity was observed in 15 cases (71.4%), and was significantly associated with RET rearrangement (P<0.001). Conclusions: The RET rearrangements were observed in 1.2% of NSCLCs. All cases of RET rearrangement were ADCs. The RET rearrangements were more likely to be observed in younger patients. Although cytoplasmic mucin production was at least focally present in 54.5% of RET-rearranged ADCs, specific histological features were not detected. PMID:24504365

  11. 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

  12. Integration of Apoptosis Signal-Regulating Kinase 1-Mediated Stress Signaling with the Akt/Protein Kinase B-IκB Kinase Cascade

    PubMed Central

    Puckett, Mary C.; Goldman, Erinn H.; Cockrell, Lisa M.; Huang, Bei; Kasinski, Andrea L.; Du, Yuhong; Wang, Cun-Yu; Lin, Anning; Ichijo, Hidenori; Khuri, Fadlo

    2013-01-01

    Cellular processes are tightly controlled through well-coordinated signaling networks that respond to conflicting cues, such as reactive oxygen species (ROS), endoplasmic reticulum (ER) stress signals, and survival factors to ensure proper cell function. We report here a direct interaction between inhibitor of κB kinase (IKK) and apoptosis signal-regulating kinase 1 (ASK1), unveiling a critical node at the junction of survival, inflammation, and stress signaling networks. IKK can be activated by growth factor stimulation or tumor necrosis factor alpha engagement. IKK forms a complex with and phosphorylates ASK1 at a sensor site, Ser967, leading to the recruitment of 14-3-3, counteracts stress signal-triggered ASK1 activation, and suppresses ASK1-mediated functions. An inhibitory role of IKK in JNK signaling has been previously reported to depend on NF-κB-mediated gene expression. Our data suggest that IKK has a dual role: a transcription-dependent and a transcription-independent action in controlling the ASK1-JNK axis, coupling IKK to ROS and ER stress response. Direct phosphorylation of ASK1 by IKK also defines a novel IKK phosphorylation motif. Because of the intimate involvement of ASK1 in diverse diseases, the IKK/ASK1 interface offers a promising target for therapeutic development. PMID:23530055

  13. Phosphoinositide kinase signaling controls ER-PM cross-talk

    PubMed Central

    Omnus, Deike J.; Manford, Andrew G.; Bader, Jakob M.; Emr, Scott D.; Stefan, Christopher J.

    2016-01-01

    Membrane lipid dynamics must be precisely regulated for normal cellular function, and disruptions in lipid homeostasis are linked to the progression of several diseases. However, little is known about the sensory mechanisms for detecting membrane composition and how lipid metabolism is regulated in response to membrane stress. We find that phosphoinositide (PI) kinase signaling controls a conserved PDK-TORC2-Akt signaling cascade as part of a homeostasis network that allows the endoplasmic reticulum (ER) to modulate essential responses, including Ca2+-regulated lipid biogenesis, upon plasma membrane (PM) stress. Furthermore, loss of ER-PM junctions impairs this protective response, leading to PM integrity defects upon heat stress. Thus PI kinase–mediated ER-PM cross-talk comprises a regulatory system that ensures cellular integrity under membrane stress conditions. PMID:26864629

  14. Extracellular signal-regulated kinases modulate capacitation of human spermatozoa.

    PubMed

    Luconi, M; Barni, T; Vannelli, G B; Krausz, C; Marra, F; Benedetti, P A; Evangelista, V; Francavilla, S; Properzi, G; Forti, G; Baldi, E

    1998-06-01

    Recent evidence indicates the presence of p21 Ras and of a protein with characteristics similar to mitogen-activated protein kinases (MAPKs), also known as extracellular signal-regulated kinases (ERKs), in mammalian spermatozoa, suggesting the occurrence of the Ras/ERK cascade in these cells. In the present study we investigated the subcellular localization of ERKs and their biological functions in human spermatozoa. Immunohistochemistry, immunofluorescence, confocal microscopy, and immunoelectron microscopy demonstrated localization of ERKs in the postacrosomal region of spermatozoa. After stimulation of acrosome reaction with the calcium ionophore A23187 and progesterone, ERKs were mostly localized at the level of the equatorial region, indicating redistribution of these proteins in acrosome-reacted spermatozoa. Two proteins of 42 and 44 kDa that are tyrosine phosphorylated in a time-dependent manner during in vitro capacitation were identified as p42 (ERK-2) and p44 (ERK-1) by means of specific antibodies. The increase in tyrosine phosphorylation of these proteins during capacitation was accompanied by increased kinase activity, as determined by the ability of ERK-1 and ERK-2 to phosphorylate the substrate myelin basic protein. The role of this activity in the occurrence of sperm capacitation was also investigated by using PD098059, an inhibitor of the MAPK cascade. The presence of this compound during in vitro capacitation inhibits ERK activation and significantly reduces the ability of spermatozoa to undergo the acrosome reaction in response to progesterone. Since only capacitated spermatozoa are able to respond to progesterone, these data strongly indicate that ERKs are involved in the regulation of capacitation. In summary, our data demonstrate the presence of functional ERKs in human spermatozoa and indicate that these enzymes are involved in activation of these cells during capacitation, providing new insight in clarifying the molecular mechanisms and the

  15. Protein kinase A activity and Hedgehog signaling pathway.

    PubMed

    Kotani, Tomoya

    2012-01-01

    Protein kinase A (PKA) is a well-known kinase that plays fundamental roles in a variety of biological processes. In Hedgehog-responsive cells, PKA plays key roles in proliferation and fate specification by modulating the transduction of Hedgehog signaling. In the absence of Hedgehog, a basal level of PKA activity represses the transcription of Hedgehog target genes. The main substrates of PKA in this process are the Ci/Gli family of bipotential transcription factors, which activate and repress Hedgehog target gene expression. PKA phosphorylates Ci/Gli, promoting the production of the repressor forms of Ci/Gli and thus repressing Hedgehog target gene expression. In contrast, the activation of Hedgehog signaling in response to Hedgehog increases the active forms of Ci/Gli, resulting in Hedgehog target gene expression. Because both decreased and increased levels of PKA activity cause abnormal cell proliferation and alter cell fate specification, the basal level of PKA activity in Hedgehog-responsive cells should be precisely regulated. However, the mechanism by which PKA activity is regulated remains obscure and appears to vary between cell types, tissues, and organisms. To date, two mechanisms have been proposed. One is a classical mechanism in which PKA activity is regulated by a small second messenger, cAMP; the other is a novel mechanism in which PKA activity is regulated by a protein, Misty somites. PMID:22391308

  16. Systematic identification of signal integration by protein kinase A

    PubMed Central

    Filteau, Marie; Diss, Guillaume; Dubé, Alexandre K.; Schraffl, Andrea; Bachmann, Verena A.; Gagnon-Arsenault, Isabelle; Chrétien, Andrée-Ève; Steunou, Anne-Lise; Dionne, Ugo; Bisson, Nicolas; Stefan, Eduard; Landry, Christian R.

    2015-01-01

    Cellular processes and homeostasis control in eukaryotic cells is achieved by the action of regulatory proteins such as protein kinase A (PKA). Although the outbound signals from PKA directed to processes such as metabolism, growth, and aging have been well charted, what regulates this conserved regulator remains to be systematically identified to understand how it coordinates biological processes. Using a yeast PKA reporter assay, we identified genes that influence PKA activity by measuring protein–protein interactions between the regulatory and the two catalytic subunits of the PKA complex in 3,726 yeast genetic-deletion backgrounds grown on two carbon sources. Overall, nearly 500 genes were found to be connected directly or indirectly to PKA regulation, including 80 core regulators, denoting a wide diversity of signals regulating PKA, within and beyond the described upstream linear pathways. PKA regulators span multiple processes, including the antagonistic autophagy and methionine biosynthesis pathways. Our results converge toward mechanisms of PKA posttranslational regulation by lysine acetylation, which is conserved between yeast and humans and that, we show, regulates protein complex formation in mammals and carbohydrate storage and aging in yeast. Taken together, these results show that the extent of PKA input matches with its output, because this kinase receives information from upstream and downstream processes, and highlight how biological processes are interconnected and coordinated by PKA. PMID:25831502

  17. Corneal Wound Healing Requires IKB kinase β Signaling in Keratocytes

    PubMed Central

    Chen, Liang; Mongan, Maureen; Meng, Qinghang; Wang, Qin; Kao, Winston; Xia, Ying

    2016-01-01

    IkB kinase β (IKKβ) is a key signaling kinase for inflammatory responses, but it also plays diverse cell type-specific roles that are not yet fully understood. Here we investigated the role of IKKβ in the cornea using IkkβΔCS mice in which the Ikkβ gene was specifically deleted in the corneal stromal keratocytes. The IkkβΔCS corneas had normal morphology, transparency and thickness; however, they did not heal well from mild alkali burn injury. In contrast to the IkkβF/F corneas that restored transparency in 2 weeks after injury, over 50% of the IkkβΔCS corneas failed to fully recover. They instead developed recurrent haze with increased stromal thickness, severe inflammation and apoptosis. This pathogenesis correlated with sustained myofibroblast transformation with increased α smooth muscle actin (α-SMA) expression, higher levels of senescence β-Gal activity and scar tissue formation at the late stage of wound healing. In addition, the IkkβΔCS corneas displayed elevated expression of hemo-oxygenase-1 (HO-1), a marker of oxidative stress, and activation of stress signaling pathways with increased JNK, c-Jun and SMAD2/3 phosphorylation. These data suggest that IKKβ in keratocytes is required to repress oxidative stress and attenuate fibrogenesis and senescence in corneal wound healing. PMID:26987064

  18. Cdc42 Regulation of Kinase Activity and Signaling by the Yeast p21-Activated Kinase Ste20

    PubMed Central

    Lamson, Rachel E.; Winters, Matthew J.; Pryciak, Peter M.

    2002-01-01

    The Saccharomyces cerevisiae kinase Ste20 is a member of the p21-activated kinase (PAK) family with several functions, including pheromone-responsive signal transduction. While PAKs are usually activated by small G proteins and Ste20 binds Cdc42, the role of Cdc42-Ste20 binding has been controversial, largely because Ste20 lacking its entire Cdc42-binding (CRIB) domain retains kinase activity and pheromone response. Here we show that, unlike CRIB deletion, point mutations in the Ste20 CRIB domain that disrupt Cdc42 binding also disrupt pheromone signaling. We also found that Ste20 kinase activity is stimulated by GTP-bound Cdc42 in vivo and this effect is blocked by the CRIB point mutations. Moreover, the Ste20 CRIB and kinase domains bind each other, and mutations that disrupt this interaction cause hyperactive kinase activity and bypass the requirement for Cdc42 binding. These observations demonstrate that the Ste20 CRIB domain is autoinhibitory and that this negative effect is antagonized by Cdc42 to promote Ste20 kinase activity and signaling. Parallel results were observed for filamentation pathway signaling, suggesting that the requirement for Cdc42-Ste20 interaction is not qualitatively different between the mating and filamentation pathways. While necessary for pheromone signaling, the role of the Cdc42-Ste20 interaction does not require regulation by pheromone or the pheromone-activated Gβγ complex, because the CRIB point mutations also disrupt signaling by activated forms of the kinase cascade scaffold protein Ste5. In total, our observations indicate that Cdc42 converts Ste20 to an active form, while pathway stimuli regulate the ability of this active Ste20 to trigger signaling through a particular pathway. PMID:11940652

  19. Casein kinase 2 dependent phosphorylation of neprilysin regulates receptor tyrosine kinase signaling to Akt.

    PubMed

    Siepmann, Martin; Kumar, Sathish; Mayer, Günter; Walter, Jochen

    2010-01-01

    Neprilysin (NEP) is a type II membrane metalloproteinase that cleaves physiologically active peptides at the cell surface thus regulating the local concentration of these peptides available for receptor binding and signal transduction. In addition, the cytoplasmic N-terminal domain of NEP interacts with the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) thereby regulating intracellular signaling via Akt. Thus, NEP serves dual functions in extracellular and intracellular signal transduction. Here, we show that NEP undergoes phosphorylation at serine residue 6 within the N-terminal cytoplasmic domain. In vitro and cell culture experiments demonstrate that Ser 6 is efficiently phosphorylated by protein kinase CK2. The phosphorylation of the cytoplasmic domain of NEP inhibits its interaction with PTEN. Interestingly, expression of a pseudophosphorylated NEP variant (Ser6Asp) abrogates the inhibitory effect of NEP on insulin/insulin-like growth factor-1 (IGF-1) stimulated activation of Akt. Thus, our data demonstrate a regulatory role of CK2 in the interaction of NEP with PTEN and insulin/IGF-1 signaling. PMID:20957047

  20. Follicle-stimulating Hormone Activates Extracellular Signal-regulated Kinase but Not Extracellular Signal-regulated Kinase Kinase through a 100-kDa Phosphotyrosine Phosphatase*

    PubMed Central

    Cottom, Joshua; Salvador, Lisa M.; Maizels, Evelyn T.; Reierstad, Scott; Park, Youngkyu; Carr, Daniel W.; Davare, Monika A.; Hell, Johannes W.; Palmer, Stephen S.; Dent, Paul; Kawakatsu, Hisaaki; Ogata, Masato; Hunzicker-Dunn, Mary

    2006-01-01

    In this report we sought to elucidate the mechanism by which the follicle-stimulating hormone (FSH) receptor signals to promote activation of the p42/p44 extracellular signal-regulated protein kinases (ERKs) in granulosa cells. Results show that the ERK kinase MEK and upstream intermediates Raf-1, Ras, Src, and L-type Ca2+ channels are already partially activated in vehicle-treated cells and that FSH does not further activate them. This tonic stimulatory pathway appears to be restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase that associates with ERK in vehicle-treated cells and promotes dephosphorylation of its regulatory Tyr residue, resulting in ERK inactivation. FSH promotes the phosphorylation of this phosphotyrosine phosphatase and its dissociation from ERK, relieving ERK from inhibition and resulting in its activation by the tonic stimulatory pathway and consequent translocation to the nucleus. Consistent with this premise, FSH-stimulated ERK activation is inhibited by the cell-permeable protein kinase A-specific inhibitor peptide Myr-PKI as well as by inhibitors of MEK, Src, a Ca2+ channel blocker, and chelation of extracellular Ca2+. These results suggest that FSH stimulates ERK activity in immature granulosa cells by relieving an inhibition imposed by a 100-kDa phosphotyrosine phosphatase. PMID:12493768

  1. Tunable Signal Processing through a Kinase Control Cycle: the IKK Signaling Node

    PubMed Central

    Behar, Marcelo; Hoffmann, Alexander

    2013-01-01

    The transcription factor NFκB, a key component of the immune system, shows intricate stimulus-specific temporal dynamics. Those dynamics are thought to play a role in controlling the physiological response to cytokines and pathogens. Biochemical evidence suggests that the NFκB inducing kinase, IKK, a signaling hub onto which many signaling pathways converge, is regulated via a regulatory cycle comprising a poised, an active, and an inactive state. We hypothesize that it operates as a modulator of signal dynamics, actively reshaping the signals generated at the receptor proximal level. Here we show that a regulatory cycle can function in at least three dynamical regimes, tunable by regulating a single kinetic parameter. In particular, the simplest three-state regulatory cycle can generate signals with two well-defined phases, each with distinct coding capabilities in terms of the information they can carry about the stimulus. We also demonstrate that such a kinase cycle can function as a signal categorizer classifying diverse incoming signals into outputs with a limited set of temporal activity profiles. Finally, we discuss the extension of the results to other regulatory motifs that could be understood in terms of the regimes of the three-state cycle. PMID:23823243

  2. Interleukin 24 is induced by the RET/PTC3 oncoprotein and is an autocrine growth factor for epithelial cells.

    PubMed

    Shinohara, Shogo; Rothstein, Jay L

    2004-09-30

    Thyroid cancers, like hematological malignancies, are commonly associated with chromosomal translocations leading to the formation of fusion proteins. Through altered signaling by fusion proteins, cell death and survival pathways are disrupted and the physiological balance of cell-cell communication may be lost. A consequence of this disruption is the release of factors by stressed cells that alert the host. One type of host response is leukocytic infiltration that may develop into chronic inflammation or autoimmune disease. Although inflammation can be associated with neoplastic tissue, the mechanism driving this process is largely unknown. Therefore, to address the mechanism of cancer inflammation we investigated the effects of an oncogene in a murine model system. A comprehensive genetic analysis revealed several soluble factors that were induced by RET/papillary thyroid carcinoma (PTC)3 gene expression including several proinflammatory cytokines, chemokines and immunologically relevant costimulatory molecules. Following a large genetic screen using RP3-expressing thyroid cells, we identified a highly abundant transcript and later identified it as interleukin 24 (Il24), a cytokine with diverse tumor suppressor and inflammatory activities. We show that RET/PTC3 induces Il24 expression in rat thyrocytes and that this expression is dependent on the signaling properties of its tyrosine kinase. Likewise, RET/PTC3 induces large amounts of Il24 following expression in murine thyrocytes, but its expression is dramatically reduced in poorly differentiated carcinomas, a finding that parallels the loss of RET/PTC3 expression. Consistent with its behavior as a tumor suppressor, the loss of Il24 coincided with the loss of RET/PTC3 in poorly differentiated mouse tumors. A functional role of Il24 in the autocrine growth/survival of RET/PTC3-expressing thyroid cells was identified helping to support its role in cellular transformation. These data suggest that the induction of

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

    DOEpatents

    Wang, Zhi-Yong; Tang, Wenqiang

    2013-09-24

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

  4. Evolutionary Adaptations of Plant AGC Kinases: From Light Signaling to Cell Polarity Regulation

    PubMed Central

    Rademacher, Eike H.; Offringa, Remko

    2012-01-01

    Signaling and trafficking over membranes involves a plethora of transmembrane proteins that control the flow of compounds or relay specific signaling events. Next to external cues, internal stimuli can modify the activity or abundance of these proteins at the plasma membrane (PM). One such regulatory mechanism is protein phosphorylation by membrane-associated kinases, several of which are AGC kinases. The AGC kinase family is one of seven kinase families that are conserved in all eukaryotic genomes. In plants evolutionary adaptations introduced specific structural changes within the AGC kinases that most likely allow modulation of kinase activity by external stimuli (e.g., light). Starting from the well-defined structural basis common to all AGC kinases we review the current knowledge on the structure-function relationship in plant AGC kinases. Nine of the 39 Arabidopsis AGC kinases have now been shown to be involved in the regulation of auxin transport. In particular, AGC kinase-mediated phosphorylation of the auxin transporters ABCB1 and ABCB19 has been shown to regulate their activity, while auxin transporters of the PIN family are located to different positions at the PM depending on their phosphorylation status, which is a result of counteracting AGC kinase and PP6 phosphatase activities. We therefore focus on regulation of AGC kinase activity in this context. Identified structural adaptations of the involved AGC kinases may provide new insight into AGC kinase functionality and demonstrate their position as central hubs in the cellular network controlling plant development and growth. PMID:23162562

  5. Centrosome-Kinase Fusions Promote Oncogenic Signaling and Disrupt Centrosome Function in Myeloproliferative Neoplasms

    PubMed Central

    Lee, Joanna Y.; Hong, Wan-Jen; Majeti, Ravindra; Stearns, Tim

    2014-01-01

    Chromosomal translocations observed in myeloproliferative neoplasms (MPNs) frequently fuse genes that encode centrosome proteins and tyrosine kinases. This causes constitutive activation of the kinase resulting in aberrant, proliferative signaling. The function of centrosome proteins in these fusions is not well understood. Among others, kinase centrosome localization and constitutive kinase dimerization are possible consequences of centrosome protein-kinase fusions. To test the relative contributions of localization and dimerization on kinase signaling, we targeted inducibly dimerizable FGFR1 to the centrosome and other subcellular locations and generated a mutant of the FOP-FGFR1 MPN fusion defective in centrosome localization. Expression in mammalian cells followed by western blot analysis revealed a significant decrease in kinase signaling upon loss of FOP-FGFR1 centrosome localization. Kinase dimerization alone resulted in phosphorylation of the FGFR1 signaling target PLCγ, however levels comparable to FOP-FGFR1 required subcellular targeting in addition to kinase dimerization. Expression of MPN fusion proteins also resulted in centrosome disruption in epithelial cells and transformed patient cells. Primary human MPN cells showed masses of modified tubulin that colocalized with centrin, Smoothened (Smo), IFT88, and Arl13b. This is distinct from acute myeloid leukemia (AML) cells, which are not associated with centrosome-kinase fusions and had normal centrosomes. Our results suggest that effective proliferative MPN signaling requires both subcellular localization and dimerization of MPN kinases, both of which may be provided by centrosome protein fusion partners. Furthermore, centrosome disruption may contribute to the MPN transformation phenotype. PMID:24658090

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

    PubMed

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

    2016-01-01

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

  7. Extracellular signal regulated kinase 5 mediates signals triggered by the novel tumor promoter palytoxin

    SciTech Connect

    Charlson, Aaron T.; Zeliadt, Nicholette A.; Wattenberg, Elizabeth V.

    2009-12-01

    Palytoxin is classified as a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type skin tumor because it does not bind to or activate protein kinase C. Palytoxin is thus a novel tool for investigating alternative signaling pathways that may affect carcinogenesis. We previously showed that palytoxin activates three major members of the mitogen activated protein kinase (MAPK) family, extracellular signal regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Here we report that palytoxin also activates another MAPK family member, called ERK5, in HeLa cells and in keratinocytes derived from initiated mouse skin (308 cells). By contrast, TPA does not activate ERK5 in these cell lines. The major cell surface receptor for palytoxin is the Na+,K+-ATPase. Accordingly, ouabain blocked the ability of palytoxin to activate ERK5. Ouabain alone did not activate ERK5. ERK5 thus represents a divergence in the signaling pathways activated by these two agents that bind to the Na+,K+-ATPase. Cycloheximide, okadaic acid, and sodium orthovanadate did not mimic the effect of palytoxin on ERK5. These results indicate that the stimulation of ERK5 by palytoxin is not simply due to inhibition of protein synthesis or inhibition of serine/threonine or tyrosine phosphatases. Therefore, the mechanism by which palytoxin activates ERK5 differs from that by which it activates ERK1/2, JNK, and p38. Finally, studies that used pharmacological inhibitors and shRNA to block ERK5 action indicate that ERK5 contributes to palytoxin-stimulated c-Fos gene expression. These results suggest that ERK5 can act as an alternative mediator for transmitting diverse tumor promoter-stimulated signals.

  8. Structural assembly of the signaling competent ERK2-RSK1 heterodimeric protein kinase complex.

    PubMed

    Alexa, Anita; Gógl, Gergő; Glatz, Gábor; Garai, Ágnes; Zeke, András; Varga, János; Dudás, Erika; Jeszenői, Norbert; Bodor, Andrea; Hetényi, Csaba; Reményi, Attila

    2015-03-01

    Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase-kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzyme's (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK→MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 "docking" groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they "readjust," whereas generic kinase domain surface contacts bring them into a catalytically competent state. PMID:25730857

  9. Targeting B-cell receptor signaling kinases in chronic lymphocytic leukemia: the promise of entospletinib

    PubMed Central

    Sharman, Jeff; Di Paolo, Julie

    2016-01-01

    The B-cell receptor signaling pathway has emerged as an important therapeutic target in chronic lymphocytic leukemia and other B-cell malignancies. Novel agents have been developed targeting the signaling enzymes spleen tyrosine kinase (SYK), Bruton’s tyrosine kinase, and phosphoinositide 3-kinase delta. This review discusses the rationale for targeting these enzymes, as well as the preclinical and clinical evidence supporting their role as therapeutic targets, with a particular focus on SYK inhibition with entospletinib. PMID:27247756

  10. Targeting B-cell receptor signaling kinases in chronic lymphocytic leukemia: the promise of entospletinib.

    PubMed

    Sharman, Jeff; Di Paolo, Julie

    2016-06-01

    The B-cell receptor signaling pathway has emerged as an important therapeutic target in chronic lymphocytic leukemia and other B-cell malignancies. Novel agents have been developed targeting the signaling enzymes spleen tyrosine kinase (SYK), Bruton's tyrosine kinase, and phosphoinositide 3-kinase delta. This review discusses the rationale for targeting these enzymes, as well as the preclinical and clinical evidence supporting their role as therapeutic targets, with a particular focus on SYK inhibition with entospletinib. PMID:27247756

  11. Extracellular signal-regulated kinases in pain of peripheral origin.

    PubMed

    White, John P M; Cibelli, Mario; Fidalgo, Antonio Rei; Nagy, Istvan

    2011-01-10

    Activation of members of the family of enzymes known as extracellular signal-regulated kinases (ERKs) is now known to be involved in the development and/or maintenance of the pain associated with many inflammatory conditions, such as herniated spinal disc pain, chronic inflammatory articular pain, and the pain associated with bladder inflammation. Moreover, ERKs are implicated in the development of neuropathic pain signs in animals which are subjected to the lumbar 5 spinal nerve ligation model and the chronic constriction injury model of neuropathic pain. The position has now been reached where all scientists working on pain subjects ought to be aware of the importance of ERKs, if only because certain of these enzymes are increasingly employed as experimental markers of nociceptive processing. Here, we introduce the reader, first, to the intracellular context in which these enzymes function. Thereafter, we consider the involvement of ERKs in mediating nociceptive signalling to the brain resulting from noxious stimuli at the periphery which will be interpreted by the brain as pain of peripheral origin. PMID:20950608

  12. Regulation of Endothelial Permeability by Src Kinase Signaling

    PubMed Central

    Hu, Guochang; Place, Aaron T.; Minshall, Richard D.

    2010-01-01

    An important function of the endothelium is to regulate the transport of liquid and solutes across the semi-permeable vascular endothelial barrier. Two cellular pathways have been identified controlling endothelial barrier function. The normally restrictive paracellular pathway, which can become “leaky” during inflammation when gaps are induced between endothelial cells at the level of adherens and tight junctional complexes, and the transcellular pathway, which transports plasma proteins the size of albumin via transcytosis in vesicle carriers originating from cell surface caveolae. During non-inflammatory conditions, caveolae-mediated transport may be the primary mechanism of vascular permeability regulation of fluid phase molecules as well as lipids, hormones, and peptides that bind avidly to albumin. Src family protein tyrosine kinases have been implicated in the upstream signaling pathways that lead to endothelial hyperpermeability through both the paracellular and transcellular pathways. Endothelial barrier dysfunction not only affects vascular homeostasis and cell metabolism, but also governs drug delivery to underlying cells and tissues. In this review of the field, we discuss the current understanding of Src signaling in regulating paracellular and transcellular endothelial permeability pathways and effects on endogenous macromolecule and drug delivery. PMID:17897637

  13. Glycogen synthase kinase 3 in Wnt signaling pathway and cancer.

    PubMed

    Tejeda-Muñoz, Nydia; Robles-Flores, Martha

    2015-12-01

    Glycogen synthase kinase 3 (GSK-3) was first discovered in 1980 as one of the key enzymes of glycogen metabolism. Since then, GSK-3 has been revealed as one of the master regulators of a diverse range of signaling pathways, including those activated by Wnts, participating in the regulation of numerous cellular functions, suggesting that its activity is tightly regulated. Numerous studies have pointed to an association of GSK-3 dysregulation with the onset and progression of human diseases, including diabetes mellitus, obesity, inflammation, neurological illnesses, and cancer. Therefore, GSK-3 is recognized as an attractive therapeutic target in multiple disorders. However, the great number of substrates that are phosphorylated by GSK-3 has raised the question of whether this limits its feasibility as a therapeutic target because of the potential disruption of many cellular processes and also by the fear that inhibition of GSK-3 may stimulate or aid in malignant transformation, as GSK-3 can phosphorylate pro-oncogenic factors. This mini review focuses on the role played by GSK-3 in Wnt signaling pathway and cancer using as model colon cancer. PMID:26600003

  14. Interaction of LRRK2 with kinase and GTPase signaling cascades

    PubMed Central

    Boon, Joon Y.; Dusonchet, Julien; Trengrove, Chelsea; Wolozin, Benjamin

    2014-01-01

    LRRK2 is a protein that interacts with a plethora of signaling molecules, but the complexity of LRRK2 function presents a challenge for understanding the role of LRRK2 in the pathophysiology of Parkinson’s disease (PD). Studies of LRRK2 using over-expression in transgenic mice have been disappointing, however, studies using invertebrate systems have yielded a much clearer picture, with clear effects of LRRK2 expression, knockdown or deletion in Caenorhabditis elegans and Drosophila on modulation of survival of dopaminergic neurons. Recent studies have begun to focus attention on particular signaling cascades that are a target of LRRK2 function. LRRK2 interacts with members of the mitogen activated protein kinase (MAPK) pathway and might regulate the pathway action by acting as a scaffold that directs the location of MAPK pathway activity, without strongly affecting the amount of MAPK pathway activity. Binding to GTPases, GTPase-activating proteins and GTPase exchange factors are another strong theme in LRRK2 biology, with LRRK2 binding to rac1, cdc42, rab5, rab7L1, endoA, RGS2, ArfGAP1, and ArhGEF7. All of these molecules appear to feed into a function output for LRRK2 that modulates cytoskeletal outgrowth and vesicular dynamics, including autophagy. These functions likely impact modulation of α-synuclein aggregation and associated toxicity eliciting the disease processes that we term PD. PMID:25071441

  15. Activity-Based Probe for Histidine Kinase Signaling

    PubMed Central

    Wilke, Kaelyn E.; Francis, Samson; Carlson, Erin E.

    2012-01-01

    Bacterial two-component systems (TCSs) are signaling pathways composed of two proteins, a histidine kinase (HK) and a response regulator (RR). Upon stimulation, the HK autophosphorylates at a conserved histidine. The phosphoryl group is subsequently transferred to an aspartate on a RR, eliciting an adaptive response, often up- or downregulation of gene expression. TCS signaling controls many functions in bacteria including development, virulence and antibiotic resistance, making the proteins involved in these systems potential therapeutic targets. Efficient methods for the profiling of HKs are currently lacking. For direct readout of HK activity, we sought to design a probe that enables detection of the phosphotransfer event; however, analysis of the phosphohistidine species is made difficult by the instability of the P-N bond. We anticipated that use of a γ-thiophosphorylated ATP analog, which would yield a thiophosphorylated histidine intermediate, could overcome this challenge. We determined that the fluorophore-conjugated probe, ATPγS-BODIPY, labels active HK proteins and is competitive for the ATP-binding site. This activity-based probe provides a new strategy for analysis of TCSs and other HK-mediated processes and will facilitate both functional studies and inhibitor identification. PMID:22606938

  16. Structural assembly of the signaling competent ERK2–RSK1 heterodimeric protein kinase complex

    PubMed Central

    Alexa, Anita; Gógl, Gergő; Glatz, Gábor; Garai, Ágnes; Zeke, András; Varga, János; Dudás, Erika; Jeszenői, Norbert; Bodor, Andrea; Hetényi, Csaba; Reményi, Attila

    2015-01-01

    Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase–kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzyme's (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK→MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 “docking” groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they “readjust,” whereas generic kinase domain surface contacts bring them into a catalytically competent state. PMID:25730857

  17. Myogenic signaling of phosphatidylinositol 3-kinase requires the serine-threonine kinase Akt/protein kinase B

    PubMed Central

    Jiang, Bing-Hua; Aoki, Masahiro; Zheng, Jenny Z.; Li, Jian; Vogt, Peter K.

    1999-01-01

    The oncogene p3k, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase), strongly activates myogenic differentiation. Inhibition of endogenous PI 3-kinase activity with the specific inhibitor LY294002, or with dominant-negative mutants of PI 3-kinase, interferes with myotube formation and with the expression of muscle-specific proteins. Here we demonstrate that a downstream target of PI 3-kinase, serine-threonine kinase Akt, plays an important role in myogenic differentiation. Expression of constitutively active forms of Akt dramatically enhances myotube formation and expression of the muscle-specific proteins MyoD, creatine kinase, myosin heavy chain, and desmin. Transdominant negative forms of Akt inhibit myotube formation and the expression of muscle-specific proteins. The inhibition of myotube formation and the reduced expression of muscle-specific proteins caused by the PI 3-kinase inhibitor LY294002 are completely reversed by constitutively active forms of Akt. Wild-type cellular Akt effects a partial reversal of LY294002-induced inhibition of myogenic differentiation. This result suggests that Akt can substitute for PI 3-kinase in the stimulation of myogenesis; Akt may be an essential downstream component of PI 3-kinase-induced muscle differentiation. PMID:10051597

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2016-01-01

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

  20. Aurora A kinase activity influences calcium signaling in kidney cells.

    PubMed

    Plotnikova, Olga V; Pugacheva, Elena N; Golemis, Erica A

    2011-06-13

    Most studies of Aurora A (AurA) describe it as a mitotic centrosomal kinase. However, we and others have recently identified AurA functions as diverse as control of ciliary resorption, cell differentiation, and cell polarity control in interphase cells. In these activities, AurA is transiently activated by noncanonical signals, including Ca(2+)-dependent calmodulin binding. These and other observations suggested that AurA might be involved in pathological conditions, such as polycystic kidney disease (PKD). In this paper, we show that AurA is abundant in normal kidney tissue but is also abnormally expressed and activated in cells lining PKD-associated renal cysts. PKD arises from mutations in the PKD1 or PKD2 genes, encoding polycystins 1 and 2 (PC1 and PC2). AurA binds, phosphorylates, and reduces the activity of PC2, a Ca(2+)-permeable nonselective cation channel and, thus, limits the amplitude of Ca(2+) release from the endoplasmic reticulum. These and other findings suggest AurA may be a relevant new biomarker or target in the therapy of PKD. PMID:21670214

  1. Protein Kinase C and Extracellular Signal-Regulated Kinase Regulate Movement, Attachment, Pairing and Egg Release in Schistosoma mansoni

    PubMed Central

    Ressurreição, Margarida; De Saram, Paulu; Kirk, Ruth S.; Rollinson, David; Emery, Aidan M.; Page, Nigel M.; Davies, Angela J.; Walker, Anthony J.

    2014-01-01

    Protein kinases C (PKCs) and extracellular signal-regulated kinases (ERKs) are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using ‘smart’ antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated) throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance. PMID:24921927

  2. Mechanisms of Ephrin Receptor Protein Kinase-Independent Signaling in Amphid Axon Guidance in Caenorhabditis elegans

    PubMed Central

    Grossman, Emily N.; Giurumescu, Claudiu A.; Chisholm, Andrew D.

    2013-01-01

    Eph receptors and their ephrin ligands are key conserved regulators of axon guidance and can function in a variety of signaling modes. Here we analyze the genetic and cellular requirements for Eph signaling in a Caenorhabditis elegans axon guidance choice point, the ventral guidance of axons in the amphid commissure. The C. elegans Eph receptor EFN-1 has both kinase-dependent and kinase-independent roles in amphid ventral guidance. Of the four C. elegans ephrins, we find that only EFN-1 has a major role in amphid axon ventral guidance, and signals in both a receptor kinase-dependent and kinase-independent manner. Analysis of EFN-1 and EFN-1 expression and tissue-specific requirements is consistent with a model in which VAB-1 acts in amphid neurons, interacting with EFN-1 expressed on surrounding cells. Unexpectedly, left-hand neurons are more strongly affected than right-hand neurons by loss of Eph signaling, indicating a previously undetected left–right asymmetry in the requirement for Eph signaling. By screening candidate genes involved in Eph signaling, we find that the Eph kinase-independent pathway involves the ABL-1 nonreceptor tyrosine kinase and possibly the phosphatidylinositol 3-kinase pathway. Overexpression of ABL-1 is sufficient to rescue EFN-1 ventral guidance defects cell autonomously. Our results reveal new aspects of Eph signaling in a single axon guidance decision in vivo. PMID:23979582

  3. Targeting Bruton's tyrosine kinase signaling as an emerging therapeutic agent of B-cell malignancies

    PubMed Central

    XIA, BING; QU, FULIAN; YUAN, TIAN; ZHANG, YIZHUO

    2015-01-01

    It is becoming increasingly evident that B-cell receptor (BCR) signaling is central to the development and function of B cells. BCR signaling has emerged as a pivotal pathway and a key driver of numerous B-cell lymphomas. Disruption of BCR signaling can be lethal to malignant B cells. Recently, kinase inhibitors that target BCR signaling have induced notable clinical responses. These inhibitors include spleen tyrosine kinase, mammalian target of rapamycin, phosphoinositide 3′-kinase and Bruton's tyrosine kinase (BTK). Ibrutinib, an oral irreversible BTK inhibitor, has emerged as a promising targeted therapy for patients with B-cell malignancies. The present review discusses the current understanding of BTK-mediated BCR signaling in the biology and pathobiology of normal and malignant B cells, and the cellular interaction with the tumor microenvironment. The data on ibrutinib in the preclinical and clinical settings is also discussed, and perspectives for the future use of ibrutinib are outlined. PMID:26788133

  4. Prognostic and Predictive Values of Subcellular Localisation of RET in Renal Clear-Cell Carcinoma

    PubMed Central

    Wang, Lei; Zhang, Yu; Gao, Yu; Fan, Yang; Chen, Luyao; Liu, Kan; Meng, Qingyu; Zhao, Chaofei; Ma, Xin

    2016-01-01

    Metastatic renal cell carcinoma (RCC) presents a poor prognosis and an unpredictable course. To date, no validated biomarkers can predict the outcome of RCC. Ongoing efforts are conducted to identify the molecular markers of RCC progression, as well as the targets for novel therapeutic approaches. RET is a tyrosine kinase receptor which has been investigated as a possible target in other cancers because it is involved in oncogenic activation. To evaluate the predictive and prognostic functions of RET in ccRCC, a tissue microarray study was conducted on 273 ccRCC patients. Results showed that both RET cytoplasmic and nuclear expression were independently associated with PFS and OS, and the combined RET cytoplasmic and nuclear statuses demonstrated that the ratio of high nuclear RET and cytoplasmic RET was the strongest predictor of both PFS and OS. The high cytoplasmic RET expression retained its independent poor prognostic value in targeted drug treated patients. The RET nuclear expression was associated with distant metastasis. Moreover, the RET nuclear expression was an independent predictor of ccRCC postoperative metastasis. In conclusion, RET may be useful in prognostication and can be used at initial diagnosis to identify patients with high potential to develop metastasis. PMID:27092013

  5. Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2.

    PubMed

    Saruhashi, Masashi; Kumar Ghosh, Totan; Arai, Kenta; Ishizaki, Yumiko; Hagiwara, Kazuya; Komatsu, Kenji; Shiwa, Yuh; Izumikawa, Keiichi; Yoshikawa, Harunori; Umezawa, Taishi; Sakata, Yoichi; Takezawa, Daisuke

    2015-11-17

    Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the moss Physcomitrella patens with reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated "ARK" (for "ABA and abiotic stress-responsive Raf-like kinase") plays an essential role in the activation of SnRK2. ARK encoded by a single gene in P. patens belongs to the family of group B3 Raf-like MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms. PMID:26540727

  6. Nuclear factor kappa B-inducing kinase and Ikappa B kinase-alpha signal skeletal muscle cell differentiation.

    PubMed

    Canicio, J; Ruiz-Lozano, P; Carrasco, M; Palacin, M; Chien, K; Zorzano, A; Kaliman, P

    2001-06-01

    Nuclear factor kappaB (NF-kappaB)-inducing kinase (NIK), IkappaB kinase (IKK)-alpha and -beta, and IkappaBalpha are common elements that signal NF-kappaB activation in response to diverse stimuli. In this study, we analyzed the role of this pathway during insulin-like growth factor II (IGF-II)-induced myoblast differentiation. L6E9 myoblasts differentiated with IGF-II showed an induction of NF-kappaB DNA-binding activity that correlated in time with the activation of IKKalpha, IKKbeta, and NIK. Moreover, the activation of IKKalpha, IKKbeta, and NIK by IGF-II was dependent on phosphatidylinositol 3-kinase, a key regulator of myogenesis. Adenoviral transduction with the IkappaBalpha(S32A/S36A) mutant severely impaired both IGF-II-dependent NF-kappaB activation and myoblast differentiation, indicating that phosphorylation of IkappaBalpha at Ser-32 and Ser-36 is an essential myogenic step. Adenoviral transfer of wild-type or kinase-deficient forms of IKKalpha or IKKbeta revealed that IKKalpha is required for IGF-II-dependent myoblast differentiation, whereas IKKbeta is not essential for this process. Finally, overexpression of kinase-proficient wild-type NIK showed that the activation of NIK is sufficient to generate signals that trigger myogenin expression and multinucleated myotube formation in the absence of IGF-II. PMID:11279241

  7. Abl Kinases Regulate HGF/Met Signaling Required for Epithelial Cell Scattering, Tubulogenesis and Motility

    PubMed Central

    Li, Ran; Knight, Jennifer F.; Park, Morag; Pendergast, Ann Marie

    2015-01-01

    Tight regulation of receptor tyrosine kinases (RTKs) is crucial for normal development and homeostasis. Dysregulation of RTKs signaling is associated with diverse pathological conditions including cancer. The Met RTK is the receptor for hepatocyte growth factor (HGF) and is dysregulated in numerous human tumors. Here we show that Abl family of non-receptor tyrosine kinases, comprised of Abl (ABL1) and Arg (ABL2), are activated downstream of the Met receptor, and that inhibition of Abl kinases dramatically suppresses HGF-induced cell scattering and tubulogenesis. We uncover a critical role for Abl kinases in the regulation of HGF/Met-dependent RhoA activation and RhoA-mediated actomyosin contractility and actin cytoskeleton remodeling in epithelial cells. Moreover, treatment of breast cancer cells with Abl inhibitors markedly decreases Met-driven cell migration and invasion. Notably, expression of a transforming mutant of the Met receptor in the mouse mammary epithelium results in hyper-activation of both Abl and Arg kinases. Together these data demonstrate that Abl kinases link Met activation to Rho signaling and Abl kinases are required for Met-dependent cell scattering, tubulogenesis, migration, and invasion. Thus, inhibition of Abl kinases might be exploited for the treatment of cancers driven by hyperactivation of HGF/Met signaling. PMID:25946048

  8. Commentary to Gorelenkova Miller and Mieyal (2015): sulfhydryl-mediated redox signaling in inflammation: role in neurodegenerative diseases.

    PubMed

    Kato, Masashi; Ninomiya, Hiromasa; Maeda, Masao; Tanaka, Natsuko; Ilmiawati, Cimi; Yoshinaga, Masafumi

    2016-04-01

    Gorelenkova Miller and Mieyal (Arch Toxicol 89(9): 1439-1467, 2015) recently published a review paper suggesting that reversible cysteine plays a key role in redox-linked signal transduction via alteration of protein function, resulting in an association with many diseases including neurodegenerative disorders. Following their suggestions, we considered the correlation between sulfhydryl-mediated redox signaling and neurodegenerative diseases by focusing on RET proteins, a protein tyrosine kinases (PTKs) potentially sited upstream of the signal transduction cascade. c-RET is the receptor for glial cell line-derived neurotrophic factor family ligands. c-RET has been reported to be involved in not only Hirschsprung disease via development of the enteric nervous system but also neurodegenerative diseases including Parkinson's disease and amyotrophic lateral sclerosis. We also showed that c-RET might be associated with hearing loss via neurodegeneration of spiral ganglion neurons in the inner ear after birth in mice and humans. Moreover, we have reported that three kinds of oxidative stress, ultraviolet light-induced stress, osmotic stress and arsenic-induced stress, modulate kinase activity of RET-PTC1 without an extracellular domain as well as c-RET by conformational change of RET protein (dimerization) via disulfide bond formation. The oxidative stresses also modulate kinase activity of RET-PTC1 with cysteine 365 (C365) replaced by alanine with promotion of dimer formation, but not with cysteine 376 (C376) replaced by alanine. Since C376 of Ret-PTC-1 or its equivalent is most highly conserved and crucial for activity in PTKs, the cysteine could be one of major targets for oxidative stresses. PMID:26780347

  9. Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2

    PubMed Central

    Saruhashi, Masashi; Kumar Ghosh, Totan; Arai, Kenta; Ishizaki, Yumiko; Hagiwara, Kazuya; Komatsu, Kenji; Shiwa, Yuh; Izumikawa, Keiichi; Yoshikawa, Harunori; Umezawa, Taishi; Sakata, Yoichi; Takezawa, Daisuke

    2015-01-01

    Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the moss Physcomitrella patens with reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated “ARK” (for “ABA and abiotic stress-responsive Raf-like kinase”) plays an essential role in the activation of SnRK2. ARK encoded by a single gene in P. patens belongs to the family of group B3 Raf-like MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms. PMID:26540727

  10. 2-Methoxystypandrone inhibits signal transducer and activator of transcription 3 and nuclear factor-κB signaling by inhibiting Janus kinase 2 and IκB kinase.

    PubMed

    Kuang, Shan; Qi, Chunting; Liu, Jiawei; Sun, Xiaoxiao; Zhang, Qing; Sima, Zhenhua; Liu, Jingli; Li, Wuguo; Yu, Qiang

    2014-04-01

    Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) or the nuclear factor-κB (NF-κB) pathway occurs frequently in cancer cells and contributes to oncogenesis. The activation of Janus kinase 2 (JAK2) and IκB kinase (IKK) are key events in STAT3 and NF-κB signaling, respectively. We have identified 2-methoxystypandrone (2-MS) from a traditional Chinese medicinal herb Polygonum cuspidatum as a novel dual inhibitor of JAK2 and IKK. 2-MS inhibits both interleukin-6-induced and constitutively-activated STAT3, as well as tumor necrosis factor-α-induced NF-κB activation. 2-MS specifically inhibits JAK and IKKβ kinase activities but has little effect on activities of other kinases tested. The inhibitory effects of 2-MS on STAT3 and NF-κB signaling can be eliminated by DTT or glutathione and can last for 4 h after a pulse treatment. Furthermore, 2-MS inhibits growth and induces death of tumor cells, particularly those with constitutively-activated STAT3 or NF-κB signaling. We propose that the natural compound 2-MS, as a potent dual inhibitor of STAT3 and NF-κB pathways, is a promising anticancer drug candidate. PMID:24450414

  11. The RET proto-oncogene: a potential target for molecular cancer therapy.

    PubMed

    Pützer, Brigitte M; Drosten, Matthias

    2004-07-01

    The inhibition of activated receptor tyrosine kinases has defined a new era of selective cancer therapy. The value of these approaches has been demonstrated for a growing number of tyrosine kinases. Gain-of-function alterations within the RET proto-oncogene are responsible for the development of medullary, as well as papillary, thyroid carcinoma and make it a candidate for the design of targeted therapies. Recently, various strategies have been used to block the activity of RET in pre-clinical models, providing evidence that RET is a potential target for a selective cancer-therapy approach, especially when considering that the inhibition of RET activity is sufficient to revert neoplastic characteristics. Although the ideal clinically useful therapeutic option has yet to be developed, successes with other selective tyrosine kinase inhibitors encourages further effort. PMID:15242684

  12. ErbB2, EphrinB1, Src Kinase and PTPN13 Signaling Complex Regulates MAP Kinase Signaling in Human Cancers

    PubMed Central

    Vermeer, Paola D.; Bell, Megan; Lee, Kimberly; Vermeer, Daniel W.; Wieking, Byrant G.; Bilal, Erhan; Bhanot, Gyan; Drapkin, Ronny I.; Ganesan, Shridar; Klingelhutz, Aloysius J.; Hendriks, Wiljan J.; Lee, John H.

    2012-01-01

    In non-cancerous cells, phosphorylated proteins exist transiently, becoming de-phosphorylated by specific phosphatases that terminate propagation of signaling pathways. In cancers, compromised phosphatase activity and/or expression occur and contribute to tumor phenotype. The non-receptor phosphatase, PTPN13, has recently been dubbed a putative tumor suppressor. It decreased expression in breast cancer correlates with decreased overall survival. Here we show that PTPN13 regulates a new signaling complex in breast cancer consisting of ErbB2, Src, and EphrinB1. To our knowledge, this signaling complex has not been previously described. Co-immunoprecipitation and localization studies demonstrate that EphrinB1, a PTPN13 substrate, interacts with ErbB2. In addition, the oncogenic V660E ErbB2 mutation enhances this interaction, while Src kinase mediates EphrinB1 phosphorylation and subsequent MAP Kinase signaling. Decreased PTPN13 function further enhances signaling. The association of oncogene kinases (ErbB2, Src), a signaling transmembrane ligand (EphrinB1) and a phosphatase tumor suppressor (PTPN13) suggest that EphrinB1 may be a relevant therapeutic target in breast cancers harboring ErbB2-activating mutations and decreased PTPN13 expression. PMID:22279592

  13. The mobility of two kinase domains in the Escherichia coli chemoreceptor array varies with signaling state

    PubMed Central

    Briegel, Ariane; Ames, Peter; Gumbart, James C.; Oikonomou, Catherine M.; Parkinson, John S.; Jensen, Grant J.

    2013-01-01

    Summary Motile bacteria sense their physical and chemical environment through highly cooperative, ordered arrays of chemoreceptors. These signaling complexes phosphorylate a response regulator which in turn governs flagellar motor reversals, driving cells towards favorable environments. The structural changes that translate chemoeffector binding into the appropriate kinase output are not known. Here, we apply high-resolution electron cryotomography to visualize mutant chemoreceptor signaling arrays in well-defined kinase activity states. The arrays were well ordered in all signaling states, with no discernible differences in receptor conformation at 2-3 nm resolution. Differences were observed, however, in a keel-like density that we identify here as CheA kinase domains P1 and P2, which are the phosphorylation site domain and the binding domain for response regulator target proteins, respectively. Mutant receptor arrays with high kinase activities all exhibited small keels and high proteolysis susceptibility, indicative of mobile P1 and P2 domains. In contrast, arrays in kinase-off signaling states exhibited a range of keel sizes. These findings confirm that chemoreceptor arrays do not undergo large structural changes during signaling, and suggest instead that kinase activity is modulated at least in part by changes in the mobility of key domains. PMID:23802570

  14. Molecular genetics of childhood papillary thyroid carcinomas after irradiation: high prevalence of RET rearrangement.

    PubMed

    Rabes, H M; Klugbauer, S

    1998-01-01

    Epidemiological studies have revealed a connection between thyroid carcinogenesis and a history of radiation. The molecular mechanisms involved are not well understood. It has been claimed that RAS, p53 or GSP mutations and RET or TRK rearrangements might play a role in adult thyroid tumors. In childhood, the thyroid gland is particularly sensitive to ionizing radiation. The reactor accident in Chernobyl provided a unique chance to study molecular genetic aberrations in a cohort of children who developed papillary thyroid carcinomas after a short latency time after exposure to high doses of radioactive iodine isotopes. According to the concepts of molecular genetic epidemiology, exposure to a specific type of irradiation might result in a typical molecular lesion. Childhood papillary thyroid tumors after Chernobyl exhibit a high prevalence of RET rearrangement as almost the only molecular alteration. The majority showed RET/PTC3 (i.e., ELE/RET rearrangements), including several subtypes. Less frequently, RET/PTC1 (i.e., H4/RET rearrangements), and a novel type (RET/PTC5, i.e., RFG5/RET) were observed. Proof of reciprocal transcripts suggests that a balanced intrachromosomal inversion leads to this rearrangement. Breakpoint analyses revealed short homologous nucleotide stretches at the fusion points. In all types of rearrangement, the RET tyrosine kinase domain becomes controlled by 5' fused regulatory sequences of ubiquitously expressed genes that display coiled-coil regions with dimerization potential. Oncogenic activation of RET is apparently due to ligand-independent constitutive ectopic RET tyrosine kinase activity. The analysis of this cohort of children with radiation-induced thyroid tumors after Chernobyl provides insights into typical molecular aberrations in relation to a specific mode of environmental exposure and may serve as a paradigm for molecular genetic epidemiology. PMID:10027005

  15. The Oncogenic Activity of RET Point Mutants for Follicular Thyroid Cells May Account for the Occurrence of Papillary Thyroid Carcinoma in Patients Affected by Familial Medullary Thyroid Carcinoma

    PubMed Central

    Melillo, Rosa Marina; Cirafici, Anna Maria; De Falco, Valentina; Bellantoni, Marie; Chiappetta, Gennaro; Fusco, Alfredo; Carlomagno, Francesca; Picascia, Antonella; Tramontano, Donatella; Tallini, Giovanni; Santoro, Massimo

    2004-01-01

    Activating germ-line point mutations in the RET receptor are responsible for multiple endocrine neoplasia type 2-associated medullary thyroid carcinoma (MTC), whereas somatic RET rearrangements are prevalent in papillary thyroid carcinomas (PTCs). Some rare kindreds, carrying point mutations in RET, are affected by both cancer types, suggesting that, under specific circumstances, point mutations in RET can drive the generation of PTC. Here we describe a family whose siblings, affected by both PTC and MTC, carried a germ-line point mutation in the RET extracellular domain, converting cysteine 634 into serine. We tested on thyroid follicular cells the transforming activity of RET(C634S), RET(K603Q), another mutant identified in a kindred with both PTC and MTC, RET(C634R) a commonly isolated allele in MEN2A, RET(M918T) responsible for MEN2B and also identified in kindreds with both PTC and MTC, and RET/PTC1 the rearranged oncogene that characterizes bona fide PTC in patients without MTC. We show that the various RET point mutants, but not wild-type RET, scored constitutive kinase activity and exerted mitogenic effects for thyroid PC Cl 3 cells, albeit at significantly lower levels compared to RET/PTC1. The low mitogenic activity of RET point mutants paralleled their reduced kinase activity compared to RET/PTC. Furthermore, RET point mutants maintained a protein domain, the intracellular juxtamembrane domain, that exerted negative effects on the mitogenic activity. In conclusion, RET point mutants can behave as dominant oncogenes for thyroid follicular cells. Their transforming activity, however, is rather modest, providing a possible explanation for the rare association of MTC with PTC. PMID:15277225

  16. Modulators of Stomatal Lineage Signal Transduction Alter Membrane Contact Sites and Reveal Specialization among ERECTA Kinases.

    PubMed

    Ho, Chin-Min Kimmy; Paciorek, Tomasz; Abrash, Emily; Bergmann, Dominique C

    2016-08-22

    Signal transduction from a cell's surface to its interior requires dedicated signaling elements and a cellular environment conducive to signal propagation. Plant development, defense, and homeostasis rely on plasma membrane receptor-like kinases to perceive endogenous and environmental signals, but little is known about their immediate downstream targets and signaling modifiers. Using genetics, biochemistry, and live-cell imaging, we show that the VAP-RELATED SUPPRESSOR OF TMM (VST) family is required for ERECTA-mediated signaling in growth and cell-fate determination and reveal a role for ERECTA-LIKE2 in modulating signaling by its sister kinases. We show that VSTs are peripheral plasma membrane proteins that can form complexes with integral ER-membrane proteins, thereby potentially influencing the organization of the membrane milieu to promote efficient and differential signaling from the ERECTA-family members to their downstream intracellular targets. PMID:27554856

  17. Focused Proteomics Revealed a Novel Rho-kinase Signaling Pathway in the Heart.

    PubMed

    Yura, Yoshimitsu; Amano, Mutsuki; Takefuji, Mikito; Bando, Tomohiro; Suzuki, Kou; Kato, Katsuhiro; Hamaguchi, Tomonari; Hasanuzzaman Shohag, Md; Takano, Tetsuya; Funahashi, Yasuhiro; Nakamuta, Shinichi; Kuroda, Keisuke; Nishioka, Tomoki; Murohara, Toyoaki; Kaibuchi, Kozo

    2016-08-23

    Protein phosphorylation plays an important role in the physiological regulation of cardiac function. Myocardial contraction and pathogenesis of cardiac diseases have been reported to be associated with adaptive or maladaptive protein phosphorylation; however, phosphorylation signaling in the heart is not fully elucidated. We recently developed a novel kinase-interacting substrate screening (KISS) method for exhaustive screening of protein kinase substrates, using mass spectrometry and affinity chromatography. First, we examined protein phosphorylation by extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), which has been relatively well studied in cardiomyocytes. The KISS method showed that ERK and PKA mediated the phosphorylation of known cardiac-substrates of each kinase such as Rps6ka1 and cTnI, respectively. Using this method, we found about 330 proteins as Rho-kinase-mediated substrates, whose substrate in cardiomyocytes is unknown. Among them, CARP/Ankrd1, a muscle ankyrin repeat protein, was confirmed as a novel Rho-kinase-mediated substrate. We also found that non-phosphorylatable form of CARP repressed cardiac hypertrophy-related gene Myosin light chain-2v (MLC-2v) promoter activity, and decreased cell size of heart derived H9c2 myoblasts more efficiently than wild type-CARP. Thus, focused proteomics enable us to reveal a novel signaling pathway in the heart. PMID:27334702

  18. RET/PTC activation in papillary thyroid carcinoma: European Journal of Endocrinology Prize Lecture.

    PubMed

    Santoro, Massimo; Melillo, Rosa Marina; Fusco, Alfredo

    2006-11-01

    Papillary thyroid carcinoma (PTC) is frequently associated with RET gene rearrangements that generate the so-called RET/PTC oncogenes. In this review, we examine the data about the mechanisms of thyroid cell transformation, activation of downstream signal transduction pathways and modulation of gene expression induced by RET/PTC. These findings have advanced our understanding of the processes underlying PTC formation and provide the basis for novel therapeutic approaches to this disease. PMID:17062879

  19. Signalling specificity of Ser/Thr protein kinases through docking-site-mediated interactions.

    PubMed Central

    Biondi, Ricardo M; Nebreda, Angel R

    2003-01-01

    Signal transduction pathways use protein kinases for the modification of protein function by phosphorylation. A major question in the field is how protein kinases achieve the specificity required to regulate multiple cellular functions. Here we review recent studies that illuminate the mechanisms used by three families of Ser/Thr protein kinases to achieve substrate specificity. These kinases rely on direct docking interactions with substrates, using sites distinct from the phospho-acceptor sequences. Docking interactions also contribute to the specificity and regulation of protein kinase activities. Mitogen-activated protein kinase (MAPK) family members can associate with and phosphorylate specific substrates by virtue of minor variations in their docking sequences. Interestingly, the same MAPK docking pocket that binds substrates also binds docking sequences of positive and negative MAPK regulators. In the case of glycogen synthase kinase 3 (GSK3), the presence of a phosphate-binding site allows docking of previously phosphorylated (primed) substrates; this docking site is also required for the mechanism of GSK3 inhibition by phosphorylation. In contrast, non-primed substrates interact with a different region of GSK3. Phosphoinositide-dependent protein kinase-1 (PDK1) contains a hydrophobic pocket that interacts with a hydrophobic motif present in all known substrates, enabling their efficient phosphorylation. Binding of the substrate hydrophobic motifs to the pocket in the kinase domain activates PDK1 and other members of the AGC family of protein kinases. Finally, the analysis of protein kinase structures indicates that the sites used for docking substrates can also bind N- and C-terminal extensions to the kinase catalytic core and participate in the regulation of its activity. PMID:12600273

  20. SorLA controls neurotrophic activity by sorting of GDNF and its receptors GFRα1 and RET.

    PubMed

    Glerup, Simon; Lume, Maria; Olsen, Ditte; Nyengaard, Jens R; Vaegter, Christian B; Gustafsen, Camilla; Christensen, Erik I; Kjolby, Mads; Hay-Schmidt, Anders; Bender, Dirk; Madsen, Peder; Saarma, Mart; Nykjaer, Anders; Petersen, Claus M

    2013-01-31

    Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor that has reached clinical trials for Parkinson's disease. GDNF binds to its coreceptor GFRα1 and signals through the transmembrane receptor tyrosine kinase RET, or RET independently through NCAM or syndecan-3. Whereas the GDNF signaling cascades are well described, cellular turnover and trafficking of GDNF and its receptors remain poorly characterized. Here, we find that SorLA acts as sorting receptor for the GDNF/GFRα1 complex, directing it from the cell surface to endosomes. Through this mechanism, GDNF is targeted to lysosomes and degraded while GFRα1 recycles, creating an efficient GDNF clearance pathway. The SorLA/GFRα1 complex further targets RET for endocytosis but not for degradation, affecting GDNF-induced neurotrophic activities. SorLA-deficient mice display elevated GDNF levels, altered dopaminergic function, marked hyperactivity, and reduced anxiety, all of which are phenotypes related to abnormal GDNF activity. Taken together, these findings establish SorLA as a critical regulator of GDNF activity in the CNS. PMID:23333276

  1. Characterization of an Engineered Src Kinase to Study Src Signaling and Biology

    PubMed Central

    Gentry, Leanna R.; Karginov, Andrei V.; Hahn, Klaus M.; Der, Channing J.

    2014-01-01

    Summary Pharmacologic inhibitors of protein kinases comprise the vast majority of approved signal transduction inhibitors for cancer treatment. An important facet of their clinical development is the identification of the key substrates critical for their driver role in cancer. One approach for substrate identification involves evaluating the phosphorylation events associated with stable expression of an activated protein kinase. Another involves genetic or pharmacologic inhibition of protein kinase expression or activity. However, both approaches are limited by the dynamic nature of signaling, complicating whether phosphorylation changes are primary or secondary activities of kinase function. We have developed rapamycin-regulated (RapR) protein kinases as molecular tools that allow for the study of spatiotemporal regulation of signaling. Here we describe the application of this technology to the Src tyrosine kinase and oncoprotein (RapR-Src). We describe how to achieve stable expression of this tool in cell lines and how to subsequently activate the tool and determine its function in signaling and morphology. PMID:26501909

  2. 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. PMID:26842916

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

    SciTech Connect

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

    2014-10-02

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

  4. Protein kinase CK2 triggers cytosolic zinc signaling pathways by phosphorylation of zinc channel ZIP7

    PubMed Central

    Taylor, Kathryn M.; Hiscox, Stephen; Nicholson, Robert I.; Hogstrand, Christer; Kille, Peter

    2012-01-01

    The transition element zinc, which has recently been identified as an intracellular second messenger, has been implicated in various signaling pathways, including those leading to cell proliferation. Zinc channels of the ZIP protein family (Solute Carrier Family 39A, SLC39A) transiently increase the cytosolic free zinc (Zn2+) concentration in response to extracellular signals. Here, we show that phosphorylation of evolutionarily conserved residues in zinc transporter ZIP7 is associated with the gated release of Zn2+ from intracellular stores, leading to activation of tyrosine kinases and the phosphorylation of AKT and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Through pharmacological manipulation, proximity assay, and mutagenesis, we identified CK2 as the kinase responsible for ZIP7 activation. Together, the present results show that eukaryotic transition element channels can be activated post-translationally by phosphorylation eliciting a cell signaling cascade. Our study links the regulated release of zinc from intracellular stores to phosphorylation of kinases involved in proliferative responses and cell migration, suggesting a functional role for ZIP7 and zinc signals for these events which are characteristic of cancerous cells. Furthermore, the interaction of ZIP7 with CK2, a kinase that is antiapoptotoc and promotes cell division, highlights the potential for ZIP7 as a target for anti-cancer drug development. PMID:22317921

  5. The Arabidopsis MAP kinase kinase 7: A crosstalk point between Auxin signaling and defense responses?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant-pathogen interaction induces a complex host response that coordinates various signaling pathways through multiple signal molecules. Besides the well-documented signal molecules salicylic acid (SA), ethylene and jasmonic acid, auxin is emerging as an important player in this response. We recent...

  6. Venus kinase receptors: prospects in signaling and biological functions of these invertebrate kinases.

    PubMed

    Dissous, Colette; Morel, Marion; Vanderstraete, Mathieu

    2014-01-01

    Venus kinase receptors (VKRs) form a family of invertebrate receptor tyrosine kinases (RTKs) initially discovered in the parasitic platyhelminth Schistosoma mansoni. VKRs are single transmembrane receptors that contain an extracellular venus fly trap structure similar to the ligand-binding domain of G protein-coupled receptors of class C, and an intracellular tyrosine kinase domain close to that of insulin receptors. VKRs are found in a large variety of invertebrates from cnidarians to echinoderms and are highly expressed in larval stages and in gonads, suggesting a role of these proteins in embryonic and larval development as well as in reproduction. VKR gene silencing could demonstrate the function of these receptors in oogenesis as well as in spermatogenesis in S. mansoni. VKRs are activated by amino acids and are highly responsive to arginine. As many other RTKs, they form dimers when activated by ligands and induce intracellular pathways involved in protein synthesis and cellular growth, such as MAPK and PI3K/Akt/S6K pathways. VKRs are not present in vertebrates or in some invertebrate species. Questions remain open about the origin of this little-known RTK family in evolution and its role in emergence and specialization of Metazoa. What is the meaning of maintenance or loss of VKR in some phyla or species in terms of development and physiological functions? The presence of VKRs in invertebrates of economical and medical importance, such as pests, vectors of pathogens, and platyhelminth parasites, and the implication of these RTKs in gametogenesis and reproduction processes are valuable reasons to consider VKRs as interesting targets in new programs for eradication/control of pests and infectious diseases, with the main advantage in the case of parasite targeting that VKR counterparts are absent from the vertebrate host kinase panel. PMID:24860549

  7. Venus Kinase Receptors: Prospects in Signaling and Biological Functions of These Invertebrate Kinases

    PubMed Central

    Dissous, Colette; Morel, Marion; Vanderstraete, Mathieu

    2014-01-01

    Venus kinase receptors (VKRs) form a family of invertebrate receptor tyrosine kinases (RTKs) initially discovered in the parasitic platyhelminth Schistosoma mansoni. VKRs are single transmembrane receptors that contain an extracellular venus fly trap structure similar to the ligand-binding domain of G protein-coupled receptors of class C, and an intracellular tyrosine kinase domain close to that of insulin receptors. VKRs are found in a large variety of invertebrates from cnidarians to echinoderms and are highly expressed in larval stages and in gonads, suggesting a role of these proteins in embryonic and larval development as well as in reproduction. VKR gene silencing could demonstrate the function of these receptors in oogenesis as well as in spermatogenesis in S. mansoni. VKRs are activated by amino acids and are highly responsive to arginine. As many other RTKs, they form dimers when activated by ligands and induce intracellular pathways involved in protein synthesis and cellular growth, such as MAPK and PI3K/Akt/S6K pathways. VKRs are not present in vertebrates or in some invertebrate species. Questions remain open about the origin of this little-known RTK family in evolution and its role in emergence and specialization of Metazoa. What is the meaning of maintenance or loss of VKR in some phyla or species in terms of development and physiological functions? The presence of VKRs in invertebrates of economical and medical importance, such as pests, vectors of pathogens, and platyhelminth parasites, and the implication of these RTKs in gametogenesis and reproduction processes are valuable reasons to consider VKRs as interesting targets in new programs for eradication/control of pests and infectious diseases, with the main advantage in the case of parasite targeting that VKR counterparts are absent from the vertebrate host kinase panel. PMID:24860549

  8. Cross talk of tyrosine kinases with the DNA damage signaling pathways.

    PubMed

    Mahajan, Kiran; Mahajan, Nupam P

    2015-12-15

    Tyrosine kinases respond to extracellular and intracellular cues by activating specific cellular signaling cascades to regulate cell cycle, growth, proliferation, differentiation and survival. Likewise, DNA damage response proteins (DDR) activated by DNA lesions or chromatin alterations recruit the DNA repair and cell cycle checkpoint machinery to restore genome integrity and cellular homeostasis. Several new examples have been uncovered in recent studies which reveal novel epigenetic and non-epigenetic mechanisms by which tyrosine kinases interact with DDR proteins to dictate cell fate, i.e. survival or apoptosis, following DNA damage. These studies reveal the ability of tyrosine kinases to directly regulate the activity of DNA repair and cell cycle check point proteins by tyrosine phosphorylation. In addition, tyrosine kinases epigenetically regulate DNA damage signaling pathways by modifying the core histones as well as chromatin modifiers at critical tyrosine residues. Thus, deregulated tyrosine kinase driven epigenomic alterations have profound implications in cancer, aging and genetic disorders. Consequently, targeting oncogenic tyrosine kinase induced epigenetic alterations has gained significant traction in overcoming cancer cell resistance to various therapies. This review discusses mechanisms by which tyrosine kinases interact with DDR pathways to regulate processes critical for maintaining genome integrity as well as clinical strategies for targeted cancer therapies. PMID:26546517

  9. Cross talk of tyrosine kinases with the DNA damage signaling pathways

    PubMed Central

    Mahajan, Kiran; Mahajan, Nupam P.

    2015-01-01

    Tyrosine kinases respond to extracellular and intracellular cues by activating specific cellular signaling cascades to regulate cell cycle, growth, proliferation, differentiation and survival. Likewise, DNA damage response proteins (DDR) activated by DNA lesions or chromatin alterations recruit the DNA repair and cell cycle checkpoint machinery to restore genome integrity and cellular homeostasis. Several new examples have been uncovered in recent studies which reveal novel epigenetic and non-epigenetic mechanisms by which tyrosine kinases interact with DDR proteins to dictate cell fate, i.e. survival or apoptosis, following DNA damage. These studies reveal the ability of tyrosine kinases to directly regulate the activity of DNA repair and cell cycle check point proteins by tyrosine phosphorylation. In addition, tyrosine kinases epigenetically regulate DNA damage signaling pathways by modifying the core histones as well as chromatin modifiers at critical tyrosine residues. Thus, deregulated tyrosine kinase driven epigenomic alterations have profound implications in cancer, aging and genetic disorders. Consequently, targeting oncogenic tyrosine kinase induced epigenetic alterations has gained significant traction in overcoming cancer cell resistance to various therapies. This review discusses mechanisms by which tyrosine kinases interact with DDR pathways to regulate processes critical for maintaining genome integrity as well as clinical strategies for targeted cancer therapies. PMID:26546517

  10. Compensatory pathways in oncogenic kinase signaling and resistance to targeted therapies: six degrees of separation.

    PubMed

    Trusolino, Livio; Bertotti, Andrea

    2012-10-01

    The efficacy of targeted therapies against mutationally activated kinases is typically limited by the engagement of growth-promoting cues that compensate for inhibition of the targeted kinase. Initial studies have highlighted the contribution of genomic alterations, functional characteristics, and signaling feedback loops--all intrinsic to cancer cells--in sustaining such substitute activities. New evidence now indicates that the relative expression of growth factor ligands produced by the tumor microenvironment can relay redundant survival pathways, which may broadly impair responsiveness to kinase inhibitors. PMID:23071031

  11. Identification of selective inhibitors of RET and comparison with current clinical candidates through development and validation of a robust screening cascade

    PubMed Central

    Watson, Amanda J.; Hopkins, Gemma V.; Hitchin, Samantha; Begum, Habiba; Jones, Stuart; Jordan, Allan; Holt, Sarah; March, H. Nikki; Newton, Rebecca; Small, Helen; Stowell, Alex; Waddell, Ian D.; Waszkowycz, Bohdan; Ogilvie, Donald J.

    2016-01-01

    RET (REarranged during Transfection) is a receptor tyrosine kinase, which plays pivotal roles in regulating cell survival, differentiation, proliferation, migration and chemotaxis. Activation of RET is a mechanism of oncogenesis in medullary thyroid carcinomas where both germline and sporadic activating somatic mutations are prevalent. At present, there are no known specific RET inhibitors in clinical development, although many potent inhibitors of RET have been opportunistically identified through selectivity profiling of compounds initially designed to target other tyrosine kinases. Vandetanib and cabozantinib, both multi-kinase inhibitors with RET activity, are approved for use in medullary thyroid carcinoma, but additional pharmacological activities, most notably inhibition of vascular endothelial growth factor - VEGFR2 (KDR), lead to dose-limiting toxicity. The recent identification of RET fusions present in ~1% of lung adenocarcinoma patients has renewed interest in the identification and development of more selective RET inhibitors lacking the toxicities associated with the current treatments. In an earlier publication [Newton et al, 2016; 1] we reported the discovery of a series of 2-substituted phenol quinazolines as potent and selective RET kinase inhibitors. Here we describe the development of the robust screening cascade which allowed the identification and advancement of this chemical series.  Furthermore we have profiled a panel of RET-active clinical compounds both to validate the cascade and to confirm that none display a RET-selective target profile. PMID:27429741

  12. β-arrestin drives MAP kinase signaling from clathrin-coated structures after GPCR dissociation

    PubMed Central

    Eichel, K.; Jullié, D.

    2016-01-01

    β-arrestins critically regulate G protein-coupled receptor (GPCR) signaling, not only 'arresting' the G protein signal but also modulating endocytosis and initiating a discrete G protein-independent signal via MAP kinase1–3. Despite enormous recent progress toward understanding biophysical aspects of arrestin function4,5, its cell biology remains relatively poorly understood. Two key tenets underlie the present dogma: (1) β-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and (2) MAP kinase activation requires endocytosis of formed GPCR - β-arrestin complexes6–9. We show here, using β1-adrenergic receptors, that β-arrestin-2 (Arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin and β-arrestin -dependent MAP kinase signal. These results demonstrate β-arrestin 'activation at a distance', after dissociating from its activating GPCR, and signaling from CCSs. We propose a β-arrestin signaling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery. PMID:26829388

  13. β-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation.

    PubMed

    Eichel, K; Jullié, D; von Zastrow, M

    2016-03-01

    β-Arrestins critically regulate G-protein-coupled receptor (GPCR) signalling, not only 'arresting' the G protein signal but also modulating endocytosis and initiating a discrete G-protein-independent signal through MAP kinase. Despite enormous recent progress towards understanding biophysical aspects of arrestin function, arrestin cell biology remains relatively poorly understood. Two key tenets underlie the prevailing current view: β-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and MAP kinase activation requires endocytosis of formed GPCR-β-arrestin complexes. We show here, using β1-adrenergic receptors, that β-arrestin-2 (arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin- and β-arrestin-dependent MAP kinase signal. These results demonstrate β-arrestin 'activation at a distance', after dissociating from its activating GPCR, and signalling from CCSs. We propose a β-arrestin signalling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery. PMID:26829388

  14. Quantitative analysis of receptor tyrosine kinase-effector coupling at functionally relevant stimulus levels.

    PubMed

    Li, Simin; Bhave, Devayani; Chow, Jennifer M; Riera, Thomas V; Schlee, Sandra; Rauch, Simone; Atanasova, Mariya; Cate, Richard L; Whitty, Adrian

    2015-04-17

    A major goal of current signaling research is to develop a quantitative understanding of how receptor activation is coupled to downstream signaling events and to functional cellular responses. Here, we measure how activation of the RET receptor tyrosine kinase on mouse neuroblastoma cells by the neurotrophin artemin (ART) is quantitatively coupled to key downstream effectors. We show that the efficiency of RET coupling to ERK and Akt depends strongly on ART concentration, and it is highest at the low (∼100 pM) ART levels required for neurite outgrowth. Quantitative discrimination between ERK and Akt pathway signaling similarly is highest at this low ART concentration. Stimulation of the cells with 100 pM ART activated RET at the rate of ∼10 molecules/cell/min, leading at 5-10 min to a transient peak of ∼150 phospho-ERK (pERK) molecules and ∼50 pAkt molecules per pRET, after which time the levels of these two signaling effectors fell by 25-50% while the pRET levels continued to slowly rise. Kinetic experiments showed that signaling effectors in different pathways respond to RET activation with different lag times, such that the balance of signal flux among the different pathways evolves over time. Our results illustrate that measurements using high, super-physiological growth factor levels can be misleading about quantitative features of receptor signaling. We propose a quantitative model describing how receptor-effector coupling efficiency links signal amplification to signal sensitization between receptor and effector, thereby providing insight into design principles underlying how receptors and their associated signaling machinery decode an extracellular signal to trigger a functional cellular outcome. PMID:25635057

  15. Protein Kinase PKN1 Represses Wnt/β-Catenin Signaling in Human Melanoma Cells*

    PubMed Central

    James, Richard G.; Bosch, Katherine A.; Kulikauskas, Rima M.; Yang, Peitzu T.; Robin, Nick C.; Toroni, Rachel A.; Biechele, Travis L.; Berndt, Jason D.; von Haller, Priska D.; Eng, Jimmy K.; Wolf-Yadlin, Alejandro; Chien, Andy J.; Moon, Randall T.

    2013-01-01

    Advances in phosphoproteomics have made it possible to monitor changes in protein phosphorylation that occur at different steps in signal transduction and have aided the identification of new pathway components. In the present study, we applied this technology to advance our understanding of the responses of melanoma cells to signaling initiated by the secreted ligand WNT3A. We started by comparing the phosphopeptide patterns of cells treated with WNT3A for different periods of time. Next, we integrated these data sets with the results from a siRNA screen that targeted protein kinases. This integration of siRNA screening and proteomics enabled us to identify four kinases that exhibit altered phosphorylation in response to WNT3A and that regulate a luciferase reporter of β-catenin-responsive transcription (β-catenin-activated reporter). We focused on one of these kinases, an atypical PKC kinase, protein kinase N1 (PKN1). Reducing the levels of PKN1 with siRNAs significantly enhances activation of β-catenin-activated reporter and increases apoptosis in melanoma cell lines. Using affinity purification followed by mass spectrometry, we then found that PKN1 is present in a protein complex with a WNT3A receptor, Frizzled 7, as well as with proteins that co-purify with Frizzled 7. These data establish that the protein kinase PKN1 inhibits Wnt/β-catenin signaling and sensitizes melanoma cells to cell death stimulated by WNT3A. PMID:24114839

  16. Protein Kinase D1 Signaling in Angiogenic Gene Expression and VEGF-Mediated Angiogenesis

    PubMed Central

    Ren, Bin

    2016-01-01

    Protein kinase D 1 (PKD-1) is a signaling kinase important in fundamental cell functions including migration, proliferation, and differentiation. PKD-1 is also a key regulator of gene expression and angiogenesis that is essential for cardiovascular development and tumor progression. Further understanding molecular aspects of PKD-1 signaling in the regulation of angiogenesis may have translational implications in obesity, cardiovascular disease, and cancer. The author will summarize and provide the insights into molecular mechanisms by which PKD-1 regulates transcriptional expression of angiogenic genes, focusing on the transcriptional regulation of CD36 by PKD-1-FoxO1 signaling axis along with the potential implications of this axis in arterial differentiation and morphogenesis. He will also discuss a new concept of dynamic balance between proangiogenic and antiangiogenic signaling in determining angiogenic switch, and stress how PKD-1 signaling regulates VEGF signaling-mediated angiogenesis. PMID:27200349

  17. Protein Kinase D1 Signaling in Angiogenic Gene Expression and VEGF-Mediated Angiogenesis.

    PubMed

    Ren, Bin

    2016-01-01

    Protein kinase D 1 (PKD-1) is a signaling kinase important in fundamental cell functions including migration, proliferation, and differentiation. PKD-1 is also a key regulator of gene expression and angiogenesis that is essential for cardiovascular development and tumor progression. Further understanding molecular aspects of PKD-1 signaling in the regulation of angiogenesis may have translational implications in obesity, cardiovascular disease, and cancer. The author will summarize and provide the insights into molecular mechanisms by which PKD-1 regulates transcriptional expression of angiogenic genes, focusing on the transcriptional regulation of CD36 by PKD-1-FoxO1 signaling axis along with the potential implications of this axis in arterial differentiation and morphogenesis. He will also discuss a new concept of dynamic balance between proangiogenic and antiangiogenic signaling in determining angiogenic switch, and stress how PKD-1 signaling regulates VEGF signaling-mediated angiogenesis. PMID:27200349

  18. Phosphatidylinositol 3-kinase mediates epidermal growth factor-induced activation of the c-Jun N-terminal kinase signaling pathway.

    PubMed Central

    Logan, S K; Falasca, M; Hu, P; Schlessinger, J

    1997-01-01

    The signaling events which mediate activation of c-Jun N-terminal kinase (JNK) are not yet well characterized. To broaden our understanding of upstream mediators which link extracellular signals to the JNK pathway, we investigated the role of phosphatidylinositol (PI) 3-kinase in epidermal growth factor (EGF)-mediated JNK activation. In this report we demonstrate that a dominant negative form of PI 3-kinase as well as the inhibitor wortmannin blocks EGF-induced JNK activation dramatically. However, wortmannin does not have an effect on JNK activation induced by UV irradiation or osmotic shock. In addition, a membrane-targeted, constitutively active PI 3-kinase (p110beta) was shown to produce in vivo products and to activate JNK, while a kinase-mutated form of this protein showed no activation. On the basis of these experiments, we propose that PI 3-kinase activity plays a role in EGF-induced JNK activation in these cells. PMID:9315636

  19. Role of Receptor Tyrosine Kinase Signaling in Renal Fibrosis

    PubMed Central

    Liu, Feng; Zhuang, Shougang

    2016-01-01

    Renal fibrosis can be induced in different renal diseases, but ultimately progresses to end stage renal disease. Although the pathophysiologic process of renal fibrosis have not been fully elucidated, it is characterized by glomerulosclerosis and/or tubular interstitial fibrosis, and is believed to be caused by the proliferation of renal inherent cells, including glomerular epithelial cells, mesangial cells, and endothelial cells, along with defective kidney repair, renal interstitial fibroblasts activation, and extracellular matrix deposition. Receptor tyrosine kinases (RTKs) regulate a variety of cell physiological processes, including metabolism, growth, differentiation, and survival. Many studies from in vitro and animal models have provided evidence that RTKs play important roles in the pathogenic process of renal fibrosis. It is also showed that tyrosine kinases inhibitors (TKIs) have anti-fibrotic effects in basic research and clinical trials. In this review, we summarize the evidence for involvement of specific RTKs in renal fibrosis process and the employment of TKIs as a therapeutic approach for renal fibrosis. PMID:27331812

  20. Role of Receptor Tyrosine Kinase Signaling in Renal Fibrosis.

    PubMed

    Liu, Feng; Zhuang, Shougang

    2016-01-01

    Renal fibrosis can be induced in different renal diseases, but ultimately progresses to end stage renal disease. Although the pathophysiologic process of renal fibrosis have not been fully elucidated, it is characterized by glomerulosclerosis and/or tubular interstitial fibrosis, and is believed to be caused by the proliferation of renal inherent cells, including glomerular epithelial cells, mesangial cells, and endothelial cells, along with defective kidney repair, renal interstitial fibroblasts activation, and extracellular matrix deposition. Receptor tyrosine kinases (RTKs) regulate a variety of cell physiological processes, including metabolism, growth, differentiation, and survival. Many studies from in vitro and animal models have provided evidence that RTKs play important roles in the pathogenic process of renal fibrosis. It is also showed that tyrosine kinases inhibitors (TKIs) have anti-fibrotic effects in basic research and clinical trials. In this review, we summarize the evidence for involvement of specific RTKs in renal fibrosis process and the employment of TKIs as a therapeutic approach for renal fibrosis. PMID:27331812

  1. GLP-1 Receptor Agonists and the Thyroid: C-Cell Effects in Mice Are Mediated via the GLP-1 Receptor and not Associated with RET Activation

    PubMed Central

    Knauf, Jeffrey A.; Gotfredsen, Carsten; Pilling, Andrew; Sjögren, Ingrid; Andersen, Søren; Andersen, Lene; Sietske de Boer, Anne; Manova, Katia; Barlas, Afsar; Vundavalli, Sushil; Nyborg, Niels C. Berg; Bjerre Knudsen, Lotte; Moelck, Anne Marie

    2012-01-01

    Liraglutide and exenatide are glucagon-like peptide receptor (GLP-1R) agonists used in the treatment of type 2 diabetes. Both molecules have been associated with the development of thyroid C-cell tumors after lifetime exposure in rodents. Previously, it has been reported that these tumors are preceded by increased plasma calcitonin and C-cell hyperplasia. We can now document that the murine C-cell effects are mediated via GLP-1R. Thus, 13 wk of continuous exposure to GLP-1R agonists was associated with marked increases in plasma calcitonin and in the incidence of C-cell hyperplasia in wild-type mice. In contrast, similar effects were not seen in GLP-1R knockout mice. Human C-cell cancer is often caused by activating mutations in the rearranged-during-transfection (RET) protooncogene. We developed an immunohistochemical method to assess RET activation in tissues. Liraglutide dosing to mice was not found to activate RET. Further evaluation of the signaling pathways demonstrated that liraglutide increased ribosomal S6, but not MAPK kinase, phosphorylation. These observations are consistent with effects of GLP-1R agonists on rodent C cells being mediated via mammalian target of rapamycin activation in a RET- and MAPK-independent manner. PMID:22234463

  2. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

    PubMed Central

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

    The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells. PMID:27226623

  3. Activation of the Extracellular Signal-Regulated Kinase Signaling Is Critical for Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation

    PubMed Central

    Li, Chen-Shuang; Zheng, Zhong; Su, Xiao-Xia; Wang, Fei; Ling, Michelle; Zou, Min; Zhou, Hong

    2016-01-01

    Human umbilical cord mesenchymal stem cells (hUCMSCs) are recognized as candidate progenitor cells for bone regeneration. However, the mechanism of hUCMSC osteogenesis remains unclear. In this study, we revealed that mitogen-activated protein kinases (MAPKs) signaling is involved in hUCMSC osteogenic differentiation in vitro. Particularly, the activation of c-Jun N-terminal kinases (JNK) and p38 signaling pathways maintained a consistent level in hUCMSCs through the entire 21-day osteogenic differentiation period. At the same time, the activation of extracellular signal-regulated kinases (ERK) signaling significantly increased from day 5, peaked at day 9, and declined thereafter. Moreover, gene profiling of osteogenic markers, alkaline phosphatase (ALP) activity measurement, and alizarin red staining demonstrated that the application of U0126, a specific inhibitor for ERK activation, completely prohibited hUCMSC osteogenic differentiation. However, when U0126 was removed from the culture at day 9, ERK activation and osteogenic differentiation of hUCMSCs were partially recovered. Together, these findings demonstrate that the activation of ERK signaling is essential for hUCMSC osteogenic differentiation, which points out the significance of ERK signaling pathway to regulate the osteogenic differentiation of hUCMSCs as an alternative cell source for bone tissue engineering. PMID:26989682

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

    PubMed Central

    Choi, Yun-Hee; Yang, Dong Joo; Kulkarni, Atul; Moh, Sang Hyun; Kim, Ki Woo

    2015-01-01

    Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH), Mycosporine-glycine (M-Gly), and Porphyra (P334) were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies. PMID:26703626

  5. A mouse model of KIF5B-RET fusion-dependent lung tumorigenesis.

    PubMed

    Saito, Motonobu; Ishigame, Teruhide; Tsuta, Koji; Kumamoto, Kensuke; Imai, Toshio; Kohno, Takashi

    2014-11-01

    Oncogenic fusion of the RET (rearranged during transfection) gene was recently identified as a novel driver gene aberration not only for the development of thyroid carcinoma but also of lung adenocarcinoma, the most frequent histological type of lung cancer. This study constructed and analyzed transgenic mice expressing KIF5B-RET, the predominant form of RET fusion gene specific for lung adenocarcinoma, under the control of the SPC (surfactant protein C) gene promoter. The mice expressed the KIF5B-RET fusion gene specifically in lung alveolar epithelial cells, and developed multiple tumors in the lungs. Treatment of the transgenic mice with vandetanib, which is a RET tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration for the treatment of thyroid carcinoma, for 8 or 20 weeks led to a marked reduction in the number of lung tumors (3.3 versus 0 and 6.5 versus 0.2 per tissue section, respectively; P < 0.01, t-test). The results suggest that the RET fusion functions as a driver for the development of lung tumors, whose growth is inhibited by RET tyrosine kinase inhibitors. PMID:25064355

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. The Pim-1 Protein Kinase Is an Important Regulator of MET Receptor Tyrosine Kinase Levels and Signaling

    PubMed Central

    Xiong, Ying; Song, Jin H.; Mahajan, Sandeep; DuPont, Rachel; McEachern, Kristen; DeAngelo, Daniel J.; Cortes, Jorge E.; Minden, Mark D.; Ebens, Allen; Mims, Alice; LaRue, Amanda C.

    2014-01-01

    MET, the receptor for hepatocyte growth factor (HGF), plays an important role in signaling normal and tumor cell migration and invasion. Here, we describe a previously unrecognized mechanism that promotes MET expression in multiple tumor cell types. The levels of the Pim-1 protein kinase show a positive correlation with the levels of MET protein in human tumor cell lines and patient-derived tumor materials. Using small interfering RNA (siRNA), Pim knockout mice, small-molecule inhibitors, and overexpression of Pim-1, we confirmed this correlation and found that Pim-1 kinase activity regulates HGF-induced tumor cell migration, invasion, and cell scattering. The novel biochemical mechanism for these effects involves the ability of Pim-1 to control the translation of MET by regulating the phosphorylation of eukaryotic initiation factor 4B (eIF4B) on S406. This targeted phosphorylation is required for the binding of eIF4B to the eIF3 translation initiation complex. Importantly, Pim-1 action was validated by the evaluation of patient blood and bone marrow from a phase I clinical trial of a Pim kinase inhibitor, AZD1208. These results suggest that Pim inhibitors may have an important role in the treatment of patients where MET is driving tumor biology. PMID:24777602

  8. Neuroblastoma Tyrosine Kinase Signaling Networks Involve FYN and LYN in Endosomes and Lipid Rafts

    PubMed Central

    Guo, Ailan; Stokes, Matthew P.; Kuehn, Emily D.; George, Lynn; Comb, Michael; Grimes, Mark L.

    2015-01-01

    Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma. PMID:25884760

  9. Intersecting roles of protein tyrosine kinase and calcium signaling during fertilization.

    PubMed

    Kinsey, William H

    2013-01-01

    The oocyte is a highly specialized cell that must respond to fertilization with a preprogrammed series of signal transduction events that establish a block to polyspermy, trigger resumption of the cell cycle and execution of a developmental program. The fertilization-induced calcium transient is a key signal that initiates the process of oocyte activation and studies over the last several years have examined the signaling pathways that act upstream and downstream of this calcium transient. Protein tyrosine kinase signaling was found to be an important component of the upstream pathways that stimulated calcium release at fertilization in oocytes from animals that fertilize externally, but a similar pathway has not been found in mammals which fertilize internally. The following review will examine the diversity of signaling in oocytes from marine invertebrates, amphibians, fish and mammals in an attempt to understand the basis for the observed differences. In addition to the pathways upstream of the fertilization-induced calcium transient, recent studies are beginning to unravel the role of protein tyrosine kinase signaling downstream of the calcium transient. The PYK2 kinase was found to respond to fertilization in the zebrafish system and seems to represent a novel component of the response of the oocyte to fertilization. The potential impact of impaired PTK signaling in oocyte quality will also be discussed. PMID:23201334

  10. Production of recombinant human apoptosis signal-regulating kinase 1 (ASK1) in Escherichia coli.

    PubMed

    Volynets, Galyna P; Gorbatiuk, Oksana B; Kukharenko, Oleksandr P; Usenko, Mariya O; Yarmoluk, Sergiy M

    2016-10-01

    Apoptosis signal-regulating kinase 1 (ASK1) is a mediator of the MAPK signaling cascade, which regulates different cellular processes including apoptosis, cell survival, and differentiation. The increased activity of ASK1 is associated with a number of human diseases and this protein kinase is considered as promising therapeutic target. In the present study, the kinase domain of human ASK1 was expressed in Escherichia coli (E. coli) in soluble form. The expression level of ASK1 was around 0.3-0.47 g per 1 L after using auto-induction protocol or IPTG induction. A one-step on column method for the efficient purification of recombinant ASK1 was performed. Our approach yields sufficient amount of recombinant ASK1, which can be used for inhibitor screening assays and different crystallographic studies. PMID:27245507

  11. Emerging roles of protein kinase CK2 in abscisic acid signaling

    PubMed Central

    Vilela, Belmiro; Pagès, Montserrat; Riera, Marta

    2015-01-01

    The phytohormone abscisic acid (ABA) regulates many aspects of plant growth and development as well as responses to multiple stresses. Post-translational modifications such as phosphorylation or ubiquitination have pivotal roles in the regulation of ABA signaling. In addition to the positive regulator sucrose non-fermenting-1 related protein kinase 2 (SnRK2), the relevance of the role of other protein kinases, such as CK2, has been recently highlighted. We have recently established that CK2 phosphorylates the maize ortholog of open stomata 1 OST1, ZmOST1, suggesting a role of CK2 phosphorylation in the control of ZmOST1 protein degradation (Vilela et al., 2015). CK2 is a pleiotropic enzyme involved in multiple developmental and stress-responsive pathways. This review summarizes recent advances that taken together suggest a prominent role of protein kinase CK2 in ABA signaling and related processes. PMID:26579189

  12. 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

  13. ACK1/TNK2 Tyrosine Kinase: Molecular Signaling and Evolving Role in Cancers

    PubMed Central

    Mahajan, Kiran; Mahajan, Nupam P.

    2014-01-01

    Deregulated tyrosine kinase signaling alters cellular homeostasis to drive cancer progression. The emergence of a non-receptor tyrosine kinase, ACK1 as an oncogenic kinase, has uncovered novel mechanisms by which tyrosine kinase signaling promotes cancer progression. While early studies focused on ACK1 (also known as activated Cdc42-associated kinase 1 or TNK2) as a cytosolic effecter of activated transmembrane receptor tyrosine kinases (RTKs), wherein it shuttles between the cytosol and the nucleus to rapidly transduce extracellular signals from the RTKs to the intracellular effectors, recent data unfold a new aspect of its functionality as an epigenetic regulator. ACK1 interacts with the Estrogen Receptor (ER)/histone demethylase KDM3A (JHDM2a) complex, modifies KDM3A by tyrosine phosphorylation to regulate transcriptional outcome at HOXA1 locus to promote the growth of tamoxifen-resistant breast cancer. It is also well established that ACK1 regulates the activity of Androgen Receptor (AR) by tyrosine phosphorylation to fuel the growth of hormone-refractory prostate cancers. Further, recent explosion in genomic sequencing has revealed recurrent ACK1 gene amplification and somatic mutations in a variety of human malignancies, providing a molecular basis for its role in neoplastic transformation. In this review, we will discuss the various facets of ACK1 signaling, including its newly uncovered epigenetic regulator function, which enables cells to bypass the blockade to major survival pathways to promote resistance to standard cancer treatments. Not surprisingly, cancer cells appear to acquire an `addiction’ to ACK1 mediated survival, particularly under stress conditions, such as growth factor deprivation or genotoxic insults or hormone deprivation. With the accelerated development of potent and selective ACK1 inhibitors, targeted treatment for cancers harboring aberrant ACK1 activity may soon become a clinical reality. PMID:25347744

  14. TEC protein tyrosine kinase is involved in the Erk signaling pathway induced by HGF

    SciTech Connect

    Li, Feifei; Jiang, Yinan; Zheng, Qiping; Yang, Xiaoming; Wang, Siying

    2011-01-07

    Research highlights: {yields} TEC is rapidly tyrosine-phosphorylated and activated by HGF-stimulation in vivo or after partial hepatectomy in mice. {yields} TEC enhances the activity of Elk and serum response element (SRE) in HGF signaling pathway in hepatocyte. {yields} TEC promotes hepatocyte proliferation through the Erk-MAPK pathway. -- Abstract: Background/aims: TEC, a member of the TEC family of non-receptor type protein tyrosine kinases, has recently been suggested to play a role in hepatocyte proliferation and liver regeneration. This study aims to investigate the putative mechanisms of TEC kinase regulation of hepatocyte differentiation, i.e. to explore which signaling pathway TEC is involved in, and how TEC is activated in hepatocyte after hepatectomy and hepatocyte growth factor (HGF) stimulation. Methods: We performed immunoprecipitation (IP) and immunoblotting (IB) to examine TEC tyrosine phosphorylation after partial hepatectomy in mice and HGF stimulation in WB F-344 hepatic cells. The TEC kinase activity was determined by in vitro kinase assay. Reporter gene assay, antisense oligonucleotide and TEC dominant negative mutant (TEC{sup KM}) were used to examine the possible signaling pathways in which TEC is involved. The cell proliferation rate was evaluated by {sup 3}H-TdR incorporation. Results: TEC phosphorylation and kinase activity were increased in 1 h after hepatectomy or HGF treatment. TEC enhanced the activity of Elk and serum response element (SRE). Inhibition of MEK1 suppressed TEC phosphorylation. Blocking TEC activity dramatically decreased the activation of Erk. Reduced TEC kinase activity also suppressed the proliferation of WB F-344 cells. These results suggest TEC is involved in the Ras-MAPK pathway and acts between MEK1 and Erk. Conclusions: TEC promotes hepatocyte proliferation and regeneration and is involved in HGF-induced Erk signaling pathway.

  15. Global Effects of Kinase Inhibitors on Signaling Networks Revealed by Quantitative Phosphoproteomics*

    PubMed Central

    Pan, Cuiping; Olsen, Jesper V.; Daub, Henrik; Mann, Matthias

    2009-01-01

    Aberrant signaling causes many diseases, and manipulating signaling pathways with kinase inhibitors has emerged as a promising area of drug research. Most kinase inhibitors target the conserved ATP-binding pocket; therefore specificity is a major concern. Proteomics has previously been used to identify the direct targets of kinase inhibitors upon affinity purification from cellular extracts. Here we introduce a complementary approach to evaluate the effects of kinase inhibitors on the entire cell signaling network. We used triple labeling SILAC (stable isotope labeling by amino acids in cell culture) to compare cellular phosphorylation levels for control, epidermal growth factor stimulus, and growth factor combined with kinase inhibitors. Of thousands of phosphopeptides, less than 10% had a response pattern indicative of targets of U0126 and SB202190, two widely used MAPK inhibitors. Interestingly, 83% of the growth factor-induced phosphorylation events were affected by either or both inhibitors, showing quantitatively that early signaling processes are predominantly transmitted through the MAPK cascades. In contrast to MAPK inhibitors, dasatinib, a clinical drug directed against BCR-ABL, which is the cause of chronic myelogenous leukemia, affected nearly 1,000 phosphopeptides. In addition to the proximal effects on ABL and its immediate targets, dasatinib broadly affected the downstream MAPK pathways. Pathway mapping of regulated sites implicated a variety of cellular functions, such as chromosome remodeling, RNA splicing, and cytoskeletal organization, some of which have been described in the literature before. Our assay is streamlined and generic and could become a useful tool in kinase drug development. PMID:19651622

  16. Identification of Extracellular Signal-regulated Kinase 1 (ERK1) Direct Substrates using Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics*

    PubMed Central

    Xue, Liang; Wang, Pengcheng; Cao, Pianpian; Zhu, Jian-kang; Tao, W. Andy

    2014-01-01

    Kinase mediated phosphorylation signaling is extensively involved in cellular functions and human diseases, and unraveling phosphorylation networks requires the identification of substrates targeted by kinases, which has remained challenging. We report here a novel proteomic strategy to identify the specificity and direct substrates of kinases by coupling phosphoproteomics with a sensitive stable isotope labeled kinase reaction. A whole cell extract was moderately dephosphorylated and subjected to in vitro kinase reaction under the condition in which 18O-ATP is the phosphate donor. The phosphorylated proteins are then isolated and identified by mass spectrometry, in which the heavy phosphate (+85.979 Da) labeled phosphopeptides reveal the kinase specificity. The in vitro phosphorylated proteins with heavy phosphates are further overlapped with in vivo kinase-dependent phosphoproteins for the identification of direct substrates with high confidence. The strategy allowed us to identify 46 phosphorylation sites on 38 direct substrates of extracellular signal-regulated kinase 1, including multiple known substrates and novel substrates, highlighting the ability of this high throughput method for direct kinase substrate screening. PMID:25022875

  17. Extracellular signal-regulated kinase and phosphoinositol-3 kinase mediate IGF-1 induced proliferation of fetal sheep cardiomyocytes.

    PubMed

    Sundgren, Nathan C; Giraud, George D; Schultz, Jess M; Lasarev, Michael R; Stork, Philip J S; Thornburg, Kent L

    2003-12-01

    Growth of the fetal heart involves cardiomyocyte enlargement, division, and maturation. Insulin-like growth factor-1 (IGF-1) is implicated in many aspects of growth and is likely to be important in developmental heart growth. IGF-1 stimulates the IGF-1 receptor (IGF1R) and downstream signaling pathways, including extracellular signal-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K). We hypothesized that IGF-1 stimulates cardiomyocyte proliferation and enlargement through stimulation of the ERK cascade and stimulates cardiomyocyte differentiation through the PI3K cascade. In vivo administration of Long R3 IGF-1 (LR3 IGF-1) did not stimulate cardiomyocyte hypertrophy but led to a decreased percentage of cells that were binucleated in vivo. In culture, LR3 IGF-1 increased myocyte bromodeoxyuridine (BrdU) uptake by three- to five-fold. The blockade of either ERK or PI3K signaling (by UO-126 or LY-294002, respectively) completely abolished BrdU uptake stimulated by LR3 IGF-1. LR3 IGF-1 did not increase footprint area, but as expected, phenylephrine stimulated an increase in binucleated cardiomyocyte size. We conclude that 1) IGF-1 through IGF1R stimulates cardiomyocyte division in vivo; hyperplastic growth is the most likely explanation of IGF-1 stimulated heart growth in vivo; 2) IGF-1 through IGF1R does not stimulate binucleation in vitro or in vivo; 3) IGF-1 through IGF1R does not stimulate hypertrophy either in vivo or in vitro; and 4) IGF-1 through IGF1R requires both ERK and PI3K signaling for proliferation of near-term fetal sheep cardiomyocytes in vitro. PMID:12947030

  18. Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis

    PubMed Central

    Hellesøy, Monica; Lorens, James B.

    2015-01-01

    The formation of new blood vessels by sprouting angiogenesis is tightly regulated by contextual cues that affect angiogeneic growth factor signaling. Both constitutive activation and loss of Akt kinase activity in endothelial cells impair angiogenesis, suggesting that Akt dynamics mediates contextual microenvironmental regulation. We explored the temporal regulation of Akt in endothelial cells during formation of capillary-like networks induced by cell–cell contact with vascular smooth muscle cells (vSMCs) and vSMC-associated VEGF. Expression of constitutively active Akt1 strongly inhibited network formation, whereas hemiphosphorylated Akt1 epi-alleles with reduced kinase activity had an intermediate inhibitory effect. Conversely, inhibition of Akt signaling did not affect endothelial cell migration or morphogenesis in vSMC cocultures that generate capillary-like structures. We found that endothelial Akt activity is transiently blocked by proteasomal degradation in the presence of SMCs during the initial phase of capillary-like structure formation. Suppressed Akt activity corresponded to the increased endothelial MAP kinase signaling that was required for angiogenic endothelial morphogenesis. These results reveal a regulatory principle by which cellular context regulates Akt protein dynamics, which determines MAP kinase signaling thresholds necessary drive a morphogenetic program during angiogenesis. PMID:26023089

  19. Electrochemical detection of protein kinase activity based on carboxypeptidase Y digestion triggered signal amplification.

    PubMed

    Yin, Huanshun; Wang, Xinxu; Guo, Yunlong; Zhou, Yunlei; Ai, Shiyun

    2015-04-15

    An effective assay method for monitoring protein kinase activity and screening inhibitors is greatly beneficial to kinase-related drug discovery, early diagnosis of diseases, and therapeutic effect evaluation. Herein, we develop a simple electrochemical method for detecting the activity of casein kinase II (CK2) based on phosphorylation against carboxypeptidase Y (CPY) digestion triggered signal amplification, where CK2 catalyzed phosphorylation event protects the substrate peptide from the digestion of CPY, maintains the repulsive force of the substrate peptide towards the redox probe, and results in a weak electrochemical signal. Whereas, without phosphorylation, the substrate peptide is digested by CPY and a strong electrochemical signal is obtained. The detection feasibility is demonstrated for the assay of CK2 activity with low detection limit of 0.047unit/mL. Moreover, the biosensor was used for the analysis of kinase inhibition. Based on the electrochemical signal dependent inhibitor concentration, the IC50 value of ellagic acid was estimated to be 39.77nM. The proposed method is also successfully applied to analyze CK2 activity in cell lysates, proving the applicability in complex biological samples. PMID:25460885

  20. Oxidative-stress-induced afterdepolarizations and calmodulin kinase II signaling.

    PubMed

    Xie, Lai-Hua; Chen, Fuhua; Karagueuzian, Hrayr S; Weiss, James N

    2009-01-01

    In the heart, oxidative stress caused by exogenous H(2)O(2) has been shown to induce early afterdepolarizations (EADs) and triggered activity by impairing Na current (I(Na)) inactivation. Because H(2)O(2) activates Ca(2+)/calmodulin kinase (CaMK)II, which also impairs I(Na) inactivation and promotes EADs, we hypothesized that CaMKII activation may be an important factor in EADs caused by oxidative stress. Using the patch-clamp and intracellular Ca (Ca(i)) imaging in Fluo-4 AM-loaded rabbit ventricular myocytes, we found that exposure to H(2)O(2) (0.2 to 1 mmol/L) for 5 to 15 minutes consistently induced EADs that were suppressed by the I(Na) blocker tetrodotoxin (10 micromol/L), as well as the I(Ca,L) blocker nifedipine. H(2)O(2) enhanced both peak and late I(Ca,L), consistent with CaMKII-mediated facilitation. By prolonging the action potential plateau and increasing Ca influx via I(Ca,L), H(2)O(2)-induced EADs were also frequently followed by DADs in response to spontaneous (ie, non-I(Ca,L)-gated) sarcoplasmic reticulum Ca release after repolarization. The CaMKII inhibitor KN-93 (1 micromol/L; n=4), but not its inactive analog KN-92 (1 micromol/L, n=5), prevented H(2)O(2)-induced EADs and DADs, and the selective CaMKII peptide inhibitor AIP (autocamtide-2-related inhibitory peptide) (2 micromol/L) significantly delayed their onset. In conclusion, H(2)O(2)-induced afterdepolarizations depend on both impaired I(Na) inactivation to reduce repolarization reserve and enhancement of I(Ca,L) to reverse repolarization, which are both facilitated by CaMKII activation. Our observations support a link between increased oxidative stress, CaMKII activation, and afterdepolarizations as triggers of lethal ventricular arrhythmias in diseased hearts. PMID:19038865

  1. Evidence that phytochrome functions as a protein kinase in plant light signalling

    PubMed Central

    Shin, Ah-Young; Han, Yun-Jeong; Baek, Ayoung; Ahn, Taeho; Kim, Soo Young; Nguyen, Thai Son; Son, Minky; Lee, Keun Woo; Shen, Yu; Song, Pill-Soon; Kim, Jeong-Il

    2016-01-01

    It has been suggested that plant phytochromes are autophosphorylating serine/threonine kinases. However, the biochemical properties and functional roles of putative phytochrome kinase activity in plant light signalling are largely unknown. Here, we describe the biochemical and functional characterization of Avena sativa phytochrome A (AsphyA) as a potential protein kinase. We provide evidence that phytochrome-interacting factors (PIFs) are phosphorylated by phytochromes in vitro. Domain mapping of AsphyA shows that the photosensory core region consisting of PAS-GAF-PHY domains in the N-terminal is required for the observed kinase activity. Moreover, we demonstrate that transgenic plants expressing mutant versions of AsphyA, which display reduced activity in in vitro kinase assays, show hyposensitive responses to far-red light. Further analysis reveals that far-red light-induced phosphorylation and degradation of PIF3 are significantly reduced in these transgenic plants. Collectively, these results suggest a positive relationship between phytochrome kinase activity and photoresponses in plants. PMID:27173885

  2. Evidence that phytochrome functions as a protein kinase in plant light signalling.

    PubMed

    Shin, Ah-Young; Han, Yun-Jeong; Baek, Ayoung; Ahn, Taeho; Kim, Soo Young; Nguyen, Thai Son; Son, Minky; Lee, Keun Woo; Shen, Yu; Song, Pill-Soon; Kim, Jeong-Il

    2016-01-01

    It has been suggested that plant phytochromes are autophosphorylating serine/threonine kinases. However, the biochemical properties and functional roles of putative phytochrome kinase activity in plant light signalling are largely unknown. Here, we describe the biochemical and functional characterization of Avena sativa phytochrome A (AsphyA) as a potential protein kinase. We provide evidence that phytochrome-interacting factors (PIFs) are phosphorylated by phytochromes in vitro. Domain mapping of AsphyA shows that the photosensory core region consisting of PAS-GAF-PHY domains in the N-terminal is required for the observed kinase activity. Moreover, we demonstrate that transgenic plants expressing mutant versions of AsphyA, which display reduced activity in in vitro kinase assays, show hyposensitive responses to far-red light. Further analysis reveals that far-red light-induced phosphorylation and degradation of PIF3 are significantly reduced in these transgenic plants. Collectively, these results suggest a positive relationship between phytochrome kinase activity and photoresponses in plants. PMID:27173885

  3. New protein kinase and protein phosphatase families mediate signal transduction in bacterial catabolite repression.

    PubMed

    Galinier, A; Kravanja, M; Engelmann, R; Hengstenberg, W; Kilhoffer, M C; Deutscher, J; Haiech, J

    1998-02-17

    Carbon catabolite repression (CCR) is the prototype of a signal transduction mechanism. In enteric bacteria, cAMP was considered to be the second messenger in CCR by playing a role reminiscent of its actions in eukaryotic cells. However, recent results suggest that CCR in Escherichia coli is mediated mainly by an inducer exclusion mechanism. In many Gram-positive bacteria, CCR is triggered by fructose-1,6-bisphosphate, which activates HPr kinase, presumed to be one of the most ancient serine protein kinases. We here report cloning of the Bacillus subtilis hprK and hprP genes and characterization of the encoded HPr kinase and P-Ser-HPr phosphatase. P-Ser-HPr phosphatase forms a new family of phosphatases together with bacterial phosphoglycolate phosphatase, yeast glycerol-3-phosphatase, and 2-deoxyglucose-6-phosphate phosphatase whereas HPr kinase represents a new family of protein kinases on its own. It does not contain the domain structure typical for eukaryotic protein kinases. Although up to now the HPr modifying/demodifying enzymes were thought to exist only in Gram-positive bacteria, a sequence comparison revealed that they also are present in several Gram-negative pathogenic bacteria. PMID:9465101

  4. RAS signalling through PI3-Kinase controls cell migration via modulation of Reelin expression

    PubMed Central

    Castellano, Esther; Molina-Arcas, Miriam; Krygowska, Agata Adelajda; East, Philip; Warne, Patricia; Nicol, Alastair; Downward, Julian

    2016-01-01

    RAS signalling through phosphoinositide 3-kinase (PI3-Kinase) has been shown to have an essential role in tumour initiation and maintenance. RAS also regulates cell motility and tumour invasiveness, but the role of direct RAS binding to PI3-Kinase in this remains uncertain. Here, we provide evidence that disruption of RAS interaction with PI3-Kinase p110α decreases cell motility and prevents activation of Rac GTPase. Analysis of gene expression in cells lacking RAS interaction with p110α reveals increased levels of the extracellular matrix glycoprotein Reelin and activation of its downstream pathway resulting in upregulation of E-cadherin expression. Induction of the Reelin/E-cadherin axis is also observed in Kras mutant lung tumours that are regressing due to blockade of RAS interaction with PI3-Kinase. Furthermore, loss of Reelin correlates with decreased survival of lung and breast cancer patients. Reelin thus plays a role in restraining RAS and PI3-kinase promotion of cell motility and potentially tumour metastasis. PMID:27071537

  5. Angiotensin II-triggered kinase signaling cascade in the central nervous system.

    PubMed

    Bali, Anjana; Jaggi, Amteshwar Singh

    2016-04-01

    Recent studies have projected the renin-angiotensin system as a central component of the physiological and pathological processes of assorted neurological disorders. Its primary effector hormone, angiotensin II (Ang II), not only mediates the physiological effects of vasoconstriction and blood pressure regulation in cardiovascular disease but is also implicated in a much wider range of neuronal activities and diseases, including Alzheimer's disease, neuronal injury, and cognitive disorders. Ang II produces different actions by acting on its two subtypes of receptors (AT1 and AT2); however, the well-known physiological actions of Ang II are mainly mediated through AT1 receptors. Moreover, recent studies also suggest the important functional role of AT2 receptor in the brain. Ang II acts on AT1 receptors and conducts its functions via MAP kinases (ERK1/2, JNK, and p38MAPK), glycogen synthase kinase, Rho/ROCK kinase, receptor tyrosine kinases (PDGF and EGFR), and nonreceptor tyrosine kinases (Src, Pyk2, and JAK/STAT). AT1R-mediated NADPH oxidase activation also leads to the generation of reactive oxygen species, widely implicated in neuroinflammation. These signaling cascades lead to glutamate excitotoxicity, apoptosis, cerebral infarction, astrocyte proliferation, nociception, neuroinflammation, and progression of other neurological disorders. The present review focuses on the Ang II-triggered signal transduction pathways in central nervous system. PMID:26574890

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

    PubMed

    Shively, Christian A; Kweon, Hye Kyong; Norman, Kaitlyn L; Mellacheruvu, Dattatreya; Xu, Tao; Sheidy, Daniel T; Dobry, Craig J; Sabath, Ivan; Cosky, Eric E P; Tran, Elizabeth J; Nesvizhskii, Alexey; Andrews, Philip C; Kumar, Anuj

    2015-10-01

    Yeast pseudohyphal filamentation is a stress-responsive growth transition relevant to processes required for virulence in pathogenic fungi. Pseudohyphal growth is controlled through a regulatory network encompassing conserved MAPK (Ste20p, Ste11p, Ste7p, Kss1p, and Fus3p), protein kinase A (Tpk2p), Elm1p, and Snf1p kinase pathways; however, the scope of these pathways is not fully understood. Here, we implemented quantitative phosphoproteomics to identify each of these signaling networks, generating a kinase-dead mutant in filamentous S. cerevisiae and surveying for differential phosphorylation. By this approach, we identified 439 phosphoproteins dependent upon pseudohyphal growth kinases. We report novel phosphorylation sites in 543 peptides, including phosphorylated residues in Ras2p and Flo8p required for wild-type filamentous growth. Phosphoproteins in these kinase signaling networks were enriched for ribonucleoprotein (RNP) granule components, and we observe co-localization of Kss1p, Fus3p, Ste20p, and Tpk2p with the RNP component Igo1p. These kinases localize in puncta with GFP-visualized mRNA, and KSS1 is required for wild-type levels of mRNA localization in RNPs. Kss1p pathway activity is reduced in lsm1Δ/Δ and pat1Δ/Δ strains, and these genes encoding P-body proteins are epistatic to STE7. The P-body protein Dhh1p is also required for hyphal development in Candida albicans. Collectively, this study presents a wealth of data identifying the yeast phosphoproteome in pseudohyphal growth and regulatory interrelationships between pseudohyphal growth kinases and RNPs. PMID:26447709

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

    PubMed Central

    Mellacheruvu, Dattatreya; Xu, Tao; Sheidy, Daniel T.; Dobry, Craig J.; Sabath, Ivan; Cosky, Eric E. P.; Tran, Elizabeth J.; Nesvizhskii, Alexey; Andrews, Philip C.; Kumar, Anuj

    2015-01-01

    Yeast pseudohyphal filamentation is a stress-responsive growth transition relevant to processes required for virulence in pathogenic fungi. Pseudohyphal growth is controlled through a regulatory network encompassing conserved MAPK (Ste20p, Ste11p, Ste7p, Kss1p, and Fus3p), protein kinase A (Tpk2p), Elm1p, and Snf1p kinase pathways; however, the scope of these pathways is not fully understood. Here, we implemented quantitative phosphoproteomics to identify each of these signaling networks, generating a kinase-dead mutant in filamentous S. cerevisiae and surveying for differential phosphorylation. By this approach, we identified 439 phosphoproteins dependent upon pseudohyphal growth kinases. We report novel phosphorylation sites in 543 peptides, including phosphorylated residues in Ras2p and Flo8p required for wild-type filamentous growth. Phosphoproteins in these kinase signaling networks were enriched for ribonucleoprotein (RNP) granule components, and we observe co-localization of Kss1p, Fus3p, Ste20p, and Tpk2p with the RNP component Igo1p. These kinases localize in puncta with GFP-visualized mRNA, and KSS1 is required for wild-type levels of mRNA localization in RNPs. Kss1p pathway activity is reduced in lsm1Δ/Δ and pat1Δ/Δ strains, and these genes encoding P-body proteins are epistatic to STE7. The P-body protein Dhh1p is also required for hyphal development in Candida albicans. Collectively, this study presents a wealth of data identifying the yeast phosphoproteome in pseudohyphal growth and regulatory interrelationships between pseudohyphal growth kinases and RNPs. PMID:26447709

  8. Rho kinase signaling pathways during stretch in primary alveolar epithelia.

    PubMed

    DiPaolo, Brian C; Margulies, Susan S

    2012-05-15

    Alveolar epithelial cells (AECs) maintain integrity of the blood-gas barrier with actin-anchored intercellular tight junctions. Stretched type I-like AECs undergo magnitude- and frequency-dependent actin cytoskeletal remodeling into perijunctional actin rings. On the basis of published studies in human pulmonary artery endothelial cells (HPAECs), we hypothesize that RhoA activity, Rho kinase (ROCK) activity, and phosphorylation of myosin light chain II (MLC2) increase in stretched type I-like AECs in a manner that is dependent on stretch magnitude, and that RhoA, ROCK, or MLC2 activity inhibition will attenuate stretch-induced actin remodeling and preserve barrier properties. Primary type I-like AEC monolayers were stretched biaxially to create a change in surface area (ΔSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min or left unstretched. Type I-like AECs were also treated with Rho pathway inhibitors (ML-7, Y-27632, or blebbistatin) and stained for F-actin or treated with the myosin phosphatase inhibitor calyculin-A and quantified for monolayer permeability. Counter to our hypothesis, ROCK activity and MLC2 phosphorylation decreased in type I-like AECs stretched to 25% and 37% ΔSA and did not change in monolayers stretched to 12% ΔSA. Furthermore, RhoA activity decreased in type I-like AECs stretched to 37% ΔSA. In contrast, MLC2 phosphorylation in HPAECs increased when HPAECs were stretched to 12% ΔSA but then decreased when they were stretched to 37% ΔSA, similar to type I-like AECs. Perijunctional actin rings were observed in unstretched type I-like AECs treated with the Rho pathway inhibitor blebbistatin. Myosin phosphatase inhibition increased MLC2 phosphorylation in stretched type I-like AECs but had no effect on monolayer permeability. In summary, stretch alters RhoA activity, ROCK activity, and MLC2 phosphorylation in a manner dependent on stretch magnitude and cell type. PMID:22287611

  9. Rho kinase signaling pathways during stretch in primary alveolar epithelia

    PubMed Central

    DiPaolo, Brian C.

    2012-01-01

    Alveolar epithelial cells (AECs) maintain integrity of the blood-gas barrier with actin-anchored intercellular tight junctions. Stretched type I-like AECs undergo magnitude- and frequency-dependent actin cytoskeletal remodeling into perijunctional actin rings. On the basis of published studies in human pulmonary artery endothelial cells (HPAECs), we hypothesize that RhoA activity, Rho kinase (ROCK) activity, and phosphorylation of myosin light chain II (MLC2) increase in stretched type I-like AECs in a manner that is dependent on stretch magnitude, and that RhoA, ROCK, or MLC2 activity inhibition will attenuate stretch-induced actin remodeling and preserve barrier properties. Primary type I-like AEC monolayers were stretched biaxially to create a change in surface area (ΔSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min or left unstretched. Type I-like AECs were also treated with Rho pathway inhibitors (ML-7, Y-27632, or blebbistatin) and stained for F-actin or treated with the myosin phosphatase inhibitor calyculin-A and quantified for monolayer permeability. Counter to our hypothesis, ROCK activity and MLC2 phosphorylation decreased in type I-like AECs stretched to 25% and 37% ΔSA and did not change in monolayers stretched to 12% ΔSA. Furthermore, RhoA activity decreased in type I-like AECs stretched to 37% ΔSA. In contrast, MLC2 phosphorylation in HPAECs increased when HPAECs were stretched to 12% ΔSA but then decreased when they were stretched to 37% ΔSA, similar to type I-like AECs. Perijunctional actin rings were observed in unstretched type I-like AECs treated with the Rho pathway inhibitor blebbistatin. Myosin phosphatase inhibition increased MLC2 phosphorylation in stretched type I-like AECs but had no effect on monolayer permeability. In summary, stretch alters RhoA activity, ROCK activity, and MLC2 phosphorylation in a manner dependent on stretch magnitude and cell type. PMID:22287611

  10. Insulin Receptor Substrate 2-mediated Phosphatidylinositol 3-kinase Signaling Selectively Inhibits Glycogen Synthase Kinase 3β to Regulate Aerobic Glycolysis*

    PubMed Central

    Landis, Justine; Shaw, Leslie M.

    2014-01-01

    Insulin receptor substrate 1 (IRS-1) and IRS-2 are cytoplasmic adaptor proteins that mediate the activation of signaling pathways in response to ligand stimulation of upstream cell surface receptors. Despite sharing a high level of homology and the ability to activate PI3K, only Irs-2 positively regulates aerobic glycolysis in mammary tumor cells. To determine the contribution of Irs-2-dependent PI3K signaling to this selective regulation, we generated an Irs-2 mutant deficient in the recruitment of PI3K. We identified four tyrosine residues (Tyr-649, Tyr-671, Tyr-734, and Tyr-814) that are essential for the association of PI3K with Irs-2 and demonstrate that combined mutation of these tyrosines inhibits glucose uptake and lactate production, two measures of aerobic glycolysis. Irs-2-dependent activation of PI3K regulates the phosphorylation of specific Akt substrates, most notably glycogen synthase kinase 3β (Gsk-3β). Inhibition of Gsk-3β by Irs-2-dependent PI3K signaling promotes glucose uptake and aerobic glycolysis. The regulation of unique subsets of Akt substrates by Irs-1 and Irs-2 may explain their non-redundant roles in mammary tumor biology. Taken together, our study reveals a novel mechanism by which Irs-2 signaling preferentially regulates tumor cell metabolism and adds to our understanding of how this adaptor protein contributes to breast cancer progression. PMID:24811175

  11. Activation of MAP kinase signaling pathway in the mussel Mytilus galloprovincialis as biomarker of environmental pollution.

    PubMed

    Châtel, A; Hamer, B; Talarmin, H; Dorange, G; Schröder, H C; Müller, W E G

    2010-03-01

    Stimulation of MAP kinase signal transduction pathway by various stressful stimuli was investigated in the marine bivalve Mytilus galloprovincialis. Analyses were performed in animals exposed in laboratory to selected pollutants and in mussels collected in winter and summer along the eastern Adriatic coast (Croatia). Effects of oxidative stress, induced by tributyltin, hydrogen peroxide and water soluble fraction of diesel fuel on the activation/phosphorylation of the three Mitogen-Activated Protein Kinases (MAPKs) p38, JNK and ERK using a newly developed ELISA procedure were evaluated. MAP kinase activation was analyzed 1h after exposure of mussels to chemical agents, and after recovery periods of 6 and 24h. Our results clearly indicated that pollutants generated different patterns of induction of the MAPK phosphorylation. Indeed, only pp38 and pJNK were activated with 11, 33 and 100 microg/L TBT, reaching a maximum activation after 6h in seawater following treatment of mussels with 11 microg/L TBT. Treatment with 0.074 and 0.222 mM H2O2 enhanced activation of both p38 and ERK. These two kinases were activated after 1h exposure, followed by a diminution after 6h of recovery in seawater and a reactivation after 24h. The levels of phosphorylated P38 and JNK were increased after mussel exposure with 7.5, 15 and 30% of water soluble fraction of diesel oil. P38 was activated concentration dependently at 1h exposure. Additionally, field study pointed out seasonal differences in MAP kinases activation as mussels collected during summer had a higher enzyme activation state than in winter, as well as sampling site differences which could be correlated to the industrial/tourism activity and environmental stresses (salinity). All the results converge towards MAP kinase signaling pathway being induced by various pollutants in M. galloprovincialis. This signaling cascade should be considered as a possible biomarker of environmental stress and pollution. PMID:19948362

  12. Retort to Religious Critics of RET.

    ERIC Educational Resources Information Center

    Nardi, Thomas J.

    This paper is concerned with people who contact clergymen for counseling who could benefit from the short-term directive therapeutic approach of Rational Emotive Therapy (RET) and the reluctance of clergymen to use RET. The integration of the precepts of Christianity and the concepts of RET is considered. This paper is specifically a response to…

  13. Sensory Protein Kinase Signaling in Schistosoma mansoni Cercariae: Host Location and Invasion.

    PubMed

    Ressurreição, Margarida; Kirk, Ruth S; Rollinson, David; Emery, Aidan M; Page, Nigel M; Walker, Anthony J

    2015-12-01

    Schistosoma mansoni cercariae display specific behavioral responses to abiotic/biotic stimuli enabling them to locate and infect the definitive human host. Here we report the effect of such stimulants on signaling pathways of cercariae in relation to host finding and invasion. Cercariae exposed to various light/temperature regimens displayed modulated protein kinase C (PKC), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) activities, with distinct responses at 37 °C and intense light/dark, when compared to 24 °C under normal light. Kinase activities were localized to regions including the oral sensory papillae, acetabular ducts, tegument, acetabular glands, and nervous system. Furthermore, linoleic acid modulated PKC and ERK activities concurrent with the temporal release of acetabular gland components. Attenuation of PKC, ERK, and p38 MAPK activities significantly reduced gland component release, particularly in response to linoleic acid, demonstrating the importance of these signaling pathways to host penetration mechanisms. PMID:26401028

  14. Protein kinase Cι expression and oncogenic signaling mechanisms in cancer.

    PubMed

    Murray, Nicole R; Kalari, Krishna R; Fields, Alan P

    2011-04-01

    Accumulating evidence demonstrates that PKCι is an oncogene and prognostic marker that is frequently targeted for genetic alteration in many major forms of human cancer. Functional data demonstrate that PKCι is required for the transformed phenotype of lung, pancreatic, ovarian, prostate, colon, and brain cancer cells. Future studies will be required to determine whether PKCι is also an oncogene in the many other cancer types that also overexpress PKCι. Studies of PKCι using genetically defined models of tumorigenesis have revealed a critical role for PKCι in multiple stages of tumorigenesis, including tumor initiation, progression, and metastasis. Recent studies in a genetic model of lung adenocarcinoma suggest a role for PKCι in transformation of lung cancer stem cells. These studies have important implications for the therapeutic use of aurothiomalate (ATM), a highly selective PKCι signaling inhibitor currently undergoing clinical evaluation. Significant progress has been made in determining the molecular mechanisms by which PKCι drives the transformed phenotype, particularly the central role played by the oncogenic PKCι-Par6 complex in transformed growth and invasion, and of several PKCι-dependent survival pathways in chemo-resistance. Future studies will be required to determine the composition and dynamics of the PKCι-Par6 complex, and the mechanisms by which oncogenic signaling through this complex is regulated. Likewise, a better understanding of the critical downstream effectors of PKCι in various human tumor types holds promise for identifying novel prognostic and surrogate markers of oncogenic PKCι activity that may be clinically useful in ongoing clinical trials of ATM. PMID:20945390

  15. The role of the Janus kinase family/signal transducer and activator of transcription signaling pathway in fibrotic renal disease

    PubMed Central

    Matsui, Futoshi; Meldrum, Kirstan K.

    2012-01-01

    Over the past several years, a number of cytokines and growth factors including transforming growth factor β1, tumor necrosis factor α, and angiotensin II have been shown to play a crucial role in renal fibrosis. The Janus kinase family (JAK) and signal transducers and activators of transcription (STATs) constitute one of the primary signaling pathways that regulate cytokine expression, and the JAK/STAT signaling pathway has increasingly been implicated in the pathophysiology of renal disease. This review examines the role of the JAK/STAT signaling pathway in fibrotic renal disease. The JAK/STAT signaling pathway is activated in a variety of renal diseases and has been implicated in the pathophysiology of renal fibrosis. Experimental evidence suggests that inhibition of the JAK/STAT signaling pathway, in particular JAK2 and STAT3, may suppress renal fibrosis and protect renal function. However, it is incompletely understood which cells activate the JAK/STAT signaling pathway and which JAK/STAT signaling pathway is activated in each renal disease. Research regarding JAK/STAT signaling and its contribution to renal disease is still ongoing in humans. Future studies are required to elucidate the potential role of JAK/STAT signaling inhibition as a therapeutic strategy in the attenuation of renal fibrosis. PMID:22883438

  16. Protein kinase B and extracellular signal-regulated kinase contribute to the chondroprotective effect of morroniside on osteoarthritis chondrocytes

    PubMed Central

    Cheng, Liang; Zeng, Guoqing; Liu, Zejun; Zhang, Bing; Cui, Xu; Zhao, Honghai; Zheng, Xinpeng; Song, Gang; Kang, Jian; Xia, Chun

    2015-01-01

    Despite extensive studies on the multifaceted roles of morroniside, the main active constituent of iridoid glycoside from Corni Fructus, the effect of morroniside on osteoarthritis (OA) chondrocytes remains poorly understood. Here, we investigated the influence of morroniside on cultured human OA chondrocytes and a rat experimental model of OA. The results showed that morroniside enhanced the cell viability and the levels of proliferating cell nuclear antigen expression (PCNA), type II collagen and aggrecan in human OA chondrocytes, indicating that morroniside promoted chondrocyte survival and matrix synthesis. Furthermore, different doses of morroniside activated protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) in human OA chondrocytes, and in turn, triggered AKT/S6 and ERK/P70S6K/S6 pathway, respectively. The PI3K/AKT inhibitor LY294002 or the MEK/ERK inhibitor U0126 attenuated the effect of morroniside on human OA chondrocytes, indicating that the activation of AKT and ERK contributed to the regulation of morroniside in human OA chondrocytes. In addition, the intra-articular injection of morroniside elevated the level of proteoglycans in cartilage matrix and the thickness of articular cartilage in a rat experimental model of OA, with the increase of AKT and ERK activation. As a consequence, morroniside has chondroprotective effect on OA chondrocytes, and may have the therapeutic potential for OA treatment. PMID:25754021

  17. Protein kinase B and extracellular signal-regulated kinase contribute to the chondroprotective effect of morroniside on osteoarthritis chondrocytes.

    PubMed

    Cheng, Liang; Zeng, Guoqing; Liu, Zejun; Zhang, Bing; Cui, Xu; Zhao, Honghai; Zheng, Xinpeng; Song, Gang; Kang, Jian; Xia, Chun

    2015-08-01

    Despite extensive studies on the multifaceted roles of morroniside, the main active constituent of iridoid glycoside from Corni Fructus, the effect of morroniside on osteoarthritis (OA) chondrocytes remains poorly understood. Here, we investigated the influence of morroniside on cultured human OA chondrocytes and a rat experimental model of OA. The results showed that morroniside enhanced the cell viability and the levels of proliferating cell nuclear antigen expression (PCNA), type II collagen and aggrecan in human OA chondrocytes, indicating that morroniside promoted chondrocyte survival and matrix synthesis. Furthermore, different doses of morroniside activated protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) in human OA chondrocytes, and in turn, triggered AKT/S6 and ERK/P70S6K/S6 pathway, respectively. The PI3K/AKT inhibitor LY294002 or the MEK/ERK inhibitor U0126 attenuated the effect of morroniside on human OA chondrocytes, indicating that the activation of AKT and ERK contributed to the regulation of morroniside in human OA chondrocytes. In addition, the intra-articular injection of morroniside elevated the level of proteoglycans in cartilage matrix and the thickness of articular cartilage in a rat experimental model of OA, with the increase of AKT and ERK activation. As a consequence, morroniside has chondroprotective effect on OA chondrocytes, and may have the therapeutic potential for OA treatment. PMID:25754021

  18. Will the Requirement by the US FDA to Simultaneously Co-Develop Companion Diagnostics (CDx) Delay the Approval of Receptor Tyrosine Kinase Inhibitors for RTK-Rearranged (ROS1-, RET-, AXL-, PDGFR-α-, NTRK1-) Non-Small Cell Lung Cancer Globally?

    PubMed

    Ou, Sai-Hong Ignatius; Soo, Ross A; Kubo, Akihito; Kawaguchi, Tomoya; Ahn, Myung-Ju

    2014-01-01

    The discovery of anaplastic lymphoma kinase (ALK) rearrangement in non-small cell lung cancer (NSCLC) in 2007 and the approval of crizotinib for the treatment of advanced ALK-rearranged NSCLC in 2011 represents a landmark in the development of targeted oncology therapy. The approval of crizotinib was accompanied simultaneously by the approval of the Vysis (Abbott Molecular) break-apart fluorescence in situ hybridization (FISH) test as the companion diagnostic (CDx) test to detect ALK rearrangement. Pfizer, the manufacturer of crizotinib, sponsored the screening of thousands of patients and the standardization of the ALK FISH test as part of the approval process for crizotinib, a first in class ALK inhibitor. Many pharmaceutical companies are now using the Food and Drug Administration (FDA)-approved ALK FISH assay to enroll patients onto trials for their own respective ALK inhibitors. In essence they are "piggybacking" on the FDA-approved ALK FISH assay without having to pay for the development of a CDx, nor screening for ALK-rearranged NSCLC patients in the protocols because screening for ALK rearrangement is now the standard of care in NSCLC after the approval of crizotinib. Since 2007, rearrangement in more receptor tyrosine kinases (RTKs) such as ROS1, RET, AXL, PDGFR-α, and NTRK1 have been discovered in NSCLC but the incidence of each subtype of RTK-rearranged NSCLC is quite rare. Crizotinib has now demonstrated significant clinical activity in ROS1-rearranged NSCLC patients. Whether crizotinib will gain official FDA approval for use in ROS1-rearranged NSCLC, on the other hand, remains unclear as there is no test for ROS1-rearrangement currently being developed to support US FDA approval as a CDx. This may be due in part to the fact that the full cost associated with the development of a pre-market approved-approved CDx must be borne by the company seeking the first drug approval in a new indication. Given the low incidence of ROS1-rearrangement in NSCLC, and

  19. The tomato kinome and the tomato kinase library ORFeome: novel resources for the study of kinases and signal transduction in tomato and solanaceae species.

    PubMed

    Singh, Dharmendra K; Calviño, Mauricio; Brauer, Elizabeth K; Fernandez-Pozo, Noe; Strickler, Susan; Yalamanchili, Roopa; Suzuki, Hideyuki; Aoki, Koh; Shibata, Daisuke; Stratmann, Johannes W; Popescu, George V; Mueller, Lukas A; Popescu, Sorina C

    2014-01-01

    Protein kinase-driven phosphorylation constitutes the core of cellular signaling. Kinase components of signal transduction pathways are often targeted for inactivation by pathogens. The study of kinases and immune signal transduction in the model crop tomato (Solanum lycopersicum) would benefit from the availability of community-wide resources for large scale and systems-level experimentation. Here, we defined the tomato kinome and performed a comprehensive comparative analysis of the tomato kinome and 15 other plant species. We constructed a tomato kinase library (TOKN 1.0) of over 300 full-length open reading frames (ORF) cloned into a recombination-based vector. We developed a high-throughput pipeline to isolate and transform tomato protoplasts. A subset of the TOKN 1.0 library kinases were expressed in planta, were purified, and were used to generate a functional tomato protein microarray. All resources created were utilized to test known and novel associations between tomato kinases and Pseudomonas syringae DC3000 effectors in a large-scale format. Bsk7 was identified as a component of the plant immune response and a candidate effector target. These resources will enable comprehensive investigations of signaling pathways and host-pathogen interactions in tomato and other Solanaceae spp. PMID:24047240

  20. GIT1/βPIX signaling proteins and PAK1 kinase regulate microtubule nucleation.

    PubMed

    Černohorská, Markéta; Sulimenko, Vadym; Hájková, Zuzana; Sulimenko, Tetyana; Sládková, Vladimíra; Vinopal, Stanislav; Dráberová, Eduarda; Dráber, Pavel

    2016-06-01

    Microtubule nucleation from γ-tubulin complexes, located at the centrosome, is an essential step in the formation of the microtubule cytoskeleton. However, the signaling mechanisms that regulate microtubule nucleation in interphase cells are largely unknown. In this study, we report that γ-tubulin is in complexes containing G protein-coupled receptor kinase-interacting protein 1 (GIT1), p21-activated kinase interacting exchange factor (βPIX), and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) in various cell lines. Immunofluorescence microscopy revealed association of GIT1, βPIX and activated PAK1 with centrosomes. Microtubule regrowth experiments showed that depletion of βPIX stimulated microtubule nucleation, while depletion of GIT1 or PAK1 resulted in decreased nucleation in the interphase cells. These data were confirmed for GIT1 and βPIX by phenotypic rescue experiments, and counting of new microtubules emanating from centrosomes during the microtubule regrowth. The importance of PAK1 for microtubule nucleation was corroborated by the inhibition of its kinase activity with IPA-3 inhibitor. GIT1 with PAK1 thus represent positive regulators, and βPIX is a negative regulator of microtubule nucleation from the interphase centrosomes. The regulatory roles of GIT1, βPIX and PAK1 in microtubule nucleation correlated with recruitment of γ-tubulin to the centrosome. Furthermore, in vitro kinase assays showed that GIT1 and βPIX, but not γ-tubulin, serve as substrates for PAK1. Finally, direct interaction of γ-tubulin with the C-terminal domain of βPIX and the N-terminal domain of GIT1, which targets this protein to the centrosome, was determined by pull-down experiments. We propose that GIT1/βPIX signaling proteins with PAK1 kinase represent a novel regulatory mechanism of microtubule nucleation in interphase cells. PMID:27012601

  1. Jelly Belly Trans-Synaptic Signaling to Anaplastic Lymphoma Kinase Regulates Neurotransmission Strength and Synapse Architecture

    PubMed Central

    Rohrbough, Jeffrey; Kent, Karla S.; Broadie, Kendal; Weiss, Joseph B.

    2012-01-01

    In Drosophila the secreted signaling molecule Jelly Belly (Jeb) activates Anaplastic Lymphoma Kinase (Alk), a receptor tyrosine kinase, in multiple developmental and adult contexts. We have shown previously that Jeb and Alk are highly enriched at Drosophila synapses within the CNS neuropil and neuromuscular junction (NMJ) and postulated a conserved intercellular signaling function. At the embryonic and larval NMJ Jeb is localized in the motor neuron presynaptic terminal whereas Alk is concentrated in the muscle postsynaptic domain surrounding boutons, consistent with anterograde trans-synaptic signaling. Here, we show by functional inhibition of Jeb-Alk signaling that neurotransmission is regulated by Jeb secretion. Jeb is a novel negative regulator of neuromuscular transmission. Reduction or inhibtion of Alk function results in enhanced synaptic transmission. Activation of Alk conversely inhibits synaptic transmission. Restoration of wildtype postsynaptic Alk expression in Alk partial loss-of-function mutants rescues NMJ transmission phenotypes and confirms that postsynaptic Alk regulates NMJ transmission. The effects of impaired Alk signaling on neurotransmission are observed in the absence of associated changes in NMJ structure. Complete removal of Jeb in motor neurons, however, disrupts both presynaptic bouton architecture and postsynaptic differentiation. Non-physiologic activation of Alk signaling also negatively regulates NMJ growth. Activation of Jeb-Alk signaling triggers the Ras-MAP kinase cascade in both pre- and postsynaptic compartments. These novel roles for Jeb-Alk signaling in the modulation of synaptic function and structure have potential implications for recently reported Alk functions in human addiction, retention of spatial memory, cognitive dysfunction in neurofibromatosis and the pathogenesis of amyotrophic lateral sclerosis. PMID:22949158

  2. The lipid kinase PIP5K1C regulates pain signaling and sensitization

    PubMed Central

    Wright, Brittany D.; Loo, Lipin; Street, Sarah E.; Ma, Anqi; Taylor-Blake, Bonnie; Stashko, Michael A.; Jin, Jian; Janzen, William P.; Frye, Stephen V.; Zylka, Mark J.

    2014-01-01

    SUMMARY Numerous pain-producing (pronociceptive) receptors signal via phosphatidylinositol 4,5- bisphosphate (PIP2) hydrolysis. However, it is currently unknown which lipid kinases generate PIP2 in nociceptive dorsal root ganglia (DRG) neurons and if these kinases regulate pronociceptive receptor signaling. Here, we found that phosphatidylinositol 4-phosphate 5 kinase type 1C (PIP5K1C) is expressed at higher levels than any other PIP5K and, based on experiments with Pip5k1c+/− mice, generates at least half of all PIP2 in DRG neurons. Additionally, Pip5k1c haploinsufficiency reduces pronociceptive receptor signaling and TRPV1 sensitization in DRG neurons as well as thermal and mechanical hypersensitivity in mouse models of chronic pain. We identified a novel small molecule inhibitor of PIP5K1C (UNC3230) in a high-throughput screen. UNC3230 lowered PIP2 levels in DRG neurons and attenuated hypersensitivity when administered intrathecally or into the hindpaw. Our studies reveal that PIP5K1C regulates PIP2- dependent nociceptive signaling and suggest that PIP5K1C is a novel therapeutic target for chronic pain. PMID:24853942

  3. Acetaldehyde alters MAP kinase signalling and epigenetic histone modifications in hepatocytes.

    PubMed

    Shukla, Shivendra D; Lee, Youn Ju; Park, Pil-hoon; Aroor, Annayya R

    2007-01-01

    Although both oxidative and non-oxidative metabolites of ethanol are involved in generating ethanol matabolic stress (Emess), the oxidative metabolite acetaldehyde plays a critical role in the cellular actions of ethanol. We have investigated the effects of acetaldehyde on p42/44 MAP kinase, p46/p54 c-jun N-terminal kinase (JNK1/JNK2) and p38 MAP kinase in hepatocytes. Acetaldehyde caused temporal activation of p42/44 MAPK followed by JNK, but the activation of the p42/44 MAPK was not a prerequisite for the JNK activation. Activation ofJNK1 by acetaldehyde was greater than JNK2. Ethanol and acetaldehyde activatedJNK have opposing roles; ethanol-induced JNK activation increased apoptosis whereas that by acetaldehyde decreased apoptosis. Acetaldehyde also caused histone H3 acetylation at Lys9 and phosphorylation of histone H3 at Serl0 and 28, the latter being dependent on p38 MAP kinase. Phosphorylation at Ser28 was higher than at Serl0. Thus acetaldehyde distinctively alters MAP kinase signalling and histone modifications, processes involved in transcriptional activation. PMID:17590997

  4. Multiple signal amplification electrogenerated chemiluminescence biosensors for sensitive protein kinase activity analysis and inhibition.

    PubMed

    Wang, Zonghua; Yan, Zhiyong; Sun, Na; Liu, Yang

    2015-06-15

    A novel electrogenerated chemiluminescence (ECL) biosensor was built for the detection of kinase activity based on multiple signal amplification nanoprobes. In this strategy, the Xanthine oxidase (XOD) and 5'-phosphate group end DNA conjugated AuNPs was integrated with the phosphorylated peptide by Zr(4+). The XOD on gold nanoparticles can catalyze dissolved oxygen to produce H2O2 in the presence of hypoxanthine (HA) which acts as a coreactor for luminol ECL reaction. In addition, due to the excellent catalytic activity of gold nanoparticle toward luminol ECL reaction and its large surface area that can accommodate large number of XOD and DNA on the surface, the ECL signal of luminol was significantly amplified, affording a highly sensitive ECL analysis of kinase activity. The as-proposed biosensor presents a low detection limit of 0.09 U mL(-1) for protein kinase A (PKA) activity, wide linear range (from 0.1 to 10 U mL(-1)) and excellent stability even in serum samples. This biosensor can also be applied for quantitative kinase inhibitor evaluation. The robust ECL biosensor provides a valuable tool for the high throughput assay in the applications of clinic diagnostic and therapeutic. PMID:25682506

  5. Tumor Phosphatidylinositol-3-Kinase Signaling and Development of Metastatic Disease in Locally Advanced Rectal Cancer

    PubMed Central

    Ree, Anne Hansen; Kristensen, Annette Torgunrud; Saelen, Marie Grøn; de Wijn, Rik; Edvardsen, Hege; Jovanovic, Jovana; Abrahamsen, Torveig Weum; Dueland, Svein; Flatmark, Kjersti

    2012-01-01

    Background Recognizing EGFR as key orchestrator of the metastatic process in colorectal cancer, but also the substantial heterogeneity of responses to anti-EGFR therapy, we examined the pattern of composite tumor kinase activities governed by EGFR-mediated signaling that might be implicated in development of metastatic disease. Patients and Methods Point mutations in KRAS, BRAF, and PIK3CA and ERBB2 amplification were determined in primary tumors from 63 patients with locally advanced rectal cancer scheduled for radical treatment. Using peptide arrays with tyrosine kinase substrates, ex vivo phosphopeptide profiles were generated from the same baseline tumor samples and correlated to metastasis-free survival. Results Unsupervised clustering analysis of the resulting phosphorylation of 102 array substrates defined two tumor classes, both consisting of cases with and without KRAS/BRAF mutations. The smaller cluster group of patients, with tumors generating high ex vivo phosphorylation of phosphatidylinositol-3-kinase-related substrates, had a particularly aggressive disease course, with almost a half of patients developing metastatic disease within one year of follow-up. Conclusion High phosphatidylinositol-3-kinase-mediated signaling activity of the primary tumor, rather than KRAS/BRAF mutation status, was identified as a hallmark of poor metastasis-free survival in patients with locally advanced rectal cancer undergoing radical treatment of the pelvic cavity. PMID:23226389

  6. Sensory Protein Kinase Signaling in Schistosoma mansoni Cercariae: Host Location and Invasion

    PubMed Central

    Ressurreição, Margarida; Kirk, Ruth S.; Rollinson, David; Emery, Aidan M.; Page, Nigel M.; Walker, Anthony J.

    2015-01-01

    Schistosoma mansoni cercariae display specific behavioral responses to abiotic/biotic stimuli enabling them to locate and infect the definitive human host. Here we report the effect of such stimulants on signaling pathways of cercariae in relation to host finding and invasion. Cercariae exposed to various light/temperature regimens displayed modulated protein kinase C (PKC), extracellular signal–regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) activities, with distinct responses at 37°C and intense light/dark, when compared to 24°C under normal light. Kinase activities were localized to regions including the oral sensory papillae, acetabular ducts, tegument, acetabular glands, and nervous system. Furthermore, linoleic acid modulated PKC and ERK activities concurrent with the temporal release of acetabular gland components. Attenuation of PKC, ERK, and p38 MAPK activities significantly reduced gland component release, particularly in response to linoleic acid, demonstrating the importance of these signaling pathways to host penetration mechanisms. PMID:26401028

  7. Human CD180 Transmits Signals via the PIM-1L Kinase

    PubMed Central

    Egli, Nicole; Zajonz, Alexandra; Burger, Matthew T.; Schweighoffer, Tamas

    2015-01-01

    Toll-like receptors (TLRs) are important sensors of the innate immune system that recognize conserved structural motifs and activate cells via a downstream signaling cascade. The CD180/MD1 molecular complex is an unusual member of the TLR family, since it lacks the components that are normally required for signal transduction by other TLRs. Therefore the CD180/MD 1 complex has been considered of being incapable of independently initiating cellular signals. Using chemogenetic approaches we identified specifically the membrane bound long form of PIM-1 kinase, PIM-1L as the mediator of CD180-dependent signaling. A dominant negative isoform of PIM-1L, but not of other PIM kinases, inhibited signaling elicited by cross-linking of CD180, and this effect was phenocopied by PIM inhibitors. PIM-1L was directed to the cell membrane by its N-terminal extension, where it colocalized and physically associated with CD180. Triggering CD180 also induced increased phosphorylation of the anti-apoptotic protein BAD in a PIM kinase-dependent fashion. Also in primary human B cells, which are the main cells expressing CD180 in man, cross-linking of CD180 by monoclonal antibodies stimulated cell survival and proliferation that was abrogated by specific inhibitors. By associating with PIM-1L, CD180 can thus obtain autonomous signaling capabilities, and this complex is then channeling inflammatory signals into B cell survival programs. Pharmacological inhibition of PIM-1 should therefore provide novel therapeutic options in diseases that respond to innate immune stimulation with subsequently increased B cell activity, such as lupus erythematosus or myasthenia gravis. PMID:26555723

  8. Human CD180 Transmits Signals via the PIM-1L Kinase.

    PubMed

    Egli, Nicole; Zajonz, Alexandra; Burger, Matthew T; Schweighoffer, Tamas

    2015-01-01

    Toll-like receptors (TLRs) are important sensors of the innate immune system that recognize conserved structural motifs and activate cells via a downstream signaling cascade. The CD180/MD1 molecular complex is an unusual member of the TLR family, since it lacks the components that are normally required for signal transduction by other TLRs. Therefore the CD180/MD 1 complex has been considered of being incapable of independently initiating cellular signals. Using chemogenetic approaches we identified specifically the membrane bound long form of PIM-1 kinase, PIM-1L as the mediator of CD180-dependent signaling. A dominant negative isoform of PIM-1L, but not of other PIM kinases, inhibited signaling elicited by cross-linking of CD180, and this effect was phenocopied by PIM inhibitors. PIM-1L was directed to the cell membrane by its N-terminal extension, where it colocalized and physically associated with CD180. Triggering CD180 also induced increased phosphorylation of the anti-apoptotic protein BAD in a PIM kinase-dependent fashion. Also in primary human B cells, which are the main cells expressing CD180 in man, cross-linking of CD180 by monoclonal antibodies stimulated cell survival and proliferation that was abrogated by specific inhibitors. By associating with PIM-1L, CD180 can thus obtain autonomous signaling capabilities, and this complex is then channeling inflammatory signals into B cell survival programs. Pharmacological inhibition of PIM-1 should therefore provide novel therapeutic options in diseases that respond to innate immune stimulation with subsequently increased B cell activity, such as lupus erythematosus or myasthenia gravis. PMID:26555723

  9. The channel-kinase TRPM7, revealing the untold story of Mg(2+) in cellular signaling.

    PubMed

    Schmitz, Carsten; Brandao, Katherine; Perraud, Anne-Laure

    2014-01-01

    Ion homeostasis dysregulations have severe effects on human health, impairing the effectiveness and appropriateness of major cellular events, including immune responses. The adverse effects of Mg(2+) deficiency on cellular physiology are well known and documented, but mechanistic insights into Mg(2+) sensitive signal transduction are still lacking. TRPM7 and its sister channel TRPM6 stand out as the only known fusions of an ion pore with a Ser/Thr kinase domain. Both channels are permeable to divalent cations and are central regulators of Mg(2+) homeostasis. One crucial aspect of TRPM7 function we have extensively studied is the relationship between its ion channel portion and its C-terminal Ser/Thr kinase domain. The modulation of ion channels by phosphorylation through exogenous kinases is common, however the covalent bound between the TRPM7 channel and its kinase suggests a novel kind of link between ion-entry and signal transduction events. Current knowledge supports a reciprocal "two-way street" model where TRPM7-kinase modulates ion transport function through Ser/Thr phosphorylation, and in turn, channel gating and ionic conditions in close proximity to the pore regulate TRPM7-kinase mediated signaling. We have shown that TRPM7 acts as a sensor of Mg(2+)-availability, adjusting key cellular functions such as the rate of cellular protein translation to the Mg(2+) nutritional status. Since molecular mechanisms controlling rates of protein translation are critical for cell growth and division in response to nutrient availability, this could have relevance for example for therapies targeted at molecules shaping the cancerous translational apparatus. In our quest to understand the biology of Mg(2+) in the context of immune responses, we found that TRPM7 associates with, and phosphorylates phospholipase C gamma 2 (PLCγ2), a pivotal molecule in the signaling pathway following B-cell receptor (BCR) activation. This contributes to the Mg(2+)-dependent modulation of

  10. MEK Kinase 2 and the Adaptor Protein Lad Regulate Extracellular Signal-Regulated Kinase 5 Activation by Epidermal Growth Factor via Src

    PubMed Central

    Sun, Weiyong; Wei, Xudong; Kesavan, Kamala; Garrington, Timothy P.; Fan, Ruihua; Mei, Junjie; Anderson, Steven M.; Gelfand, Erwin W.; Johnson, Gary L.

    2003-01-01

    Lad is an SH2 domain-containing adaptor protein that binds MEK kinase 2 (MEKK2), a mitogen-activated protein kinase (MAPK) kinase kinase for the extracellular signal-regulated kinase 5 (ERK5) and JNK pathways. Lad and MEKK2 are in a complex in resting cells. Antisense knockdown of Lad expression and targeted gene disruption of MEKK2 expression results in loss of epidermal growth factor (EGF) and stress stimuli-induced activation of ERK5. Activation of MEKK2 and the ERK5 pathway by EGF and stress stimuli is dependent on Src kinase activity. The Lad-binding motif is encoded within amino acids 228 to 282 in the N terminus of MEKK2, and expression of this motif blocks Lad-MEKK2 interaction, resulting in inhibition of Src-dependent activation of MEKK2 and ERK5. JNK activation by EGF is similarly inhibited by loss of Lad or MEKK2 expression and by blocking the interaction of MEKK2 and Lad. Our studies demonstrate that Src kinase activity is required for ERK5 activation in response to EGF, MEKK2 expression is required for ERK5 activation by Src, Lad and MEKK2 association is required for Src activation of ERK5, and EGF and Src stimulation of ERK5-regulated MEF2-dependent promoter activity requires a functional Lad-MEKK2 signaling complex. PMID:12640115

  11. Recently emerging signaling landscape of ataxia-telangiectasia mutated (ATM) kinase.

    PubMed

    Farooqi, Ammad Ahmad; Attar, Rukset; Arslan, Belkis Atasever; Romero, Mirna Azalea; ul Haq, Muhammad Fahim; Qadir, Muhammad Imran

    2014-01-01

    Research over the years has progressively and sequentially provided near complete resolution of regulators of the DNA repair pathways which are so important for cancer prevention. Ataxia-telangiectasia mutated kinase (ATM), a high-molecular-weight PI3K-family kinase has emerged as a master regulator of DNA damage signaling and extensive cross-talk between ATM and downstream proteins forms an interlaced signaling network. There is rapidly growing scientific evidence emphasizing newly emerging paradigms in ATM biology. In this review, we provide latest information regarding how oxidative stress induced activation of ATM can be utilized as a therapeutic target in different cancer cell lines and in xenografted mice. Moreover, crosstalk between autophagy and ATM is also discussed with focus on how autophagy inhibition induces apoptosis in cancer cells. PMID:25169474

  12. Propranolol Improves Impaired Hepatic Phosphatidylinositol 3-Kinase/Akt Signaling after Burn Injury

    PubMed Central

    Brooks, Natasha C; Song, Juquan; Boehning, Darren; Kraft, Robert; Finnerty, Celeste C; Herndon, David N; Jeschke, Marc G

    2012-01-01

    Severe burn injury is associated with induction of the hepatic endoplasmic reticulum (ER) stress response. ER stress leads to activation of c-Jun N-terminal kinase (JNK), suppression of insulin receptor signaling via phosphorylation of insulin receptor substrate 1 and subsequent insulin resistance. Marked and sustained increases in catecholamines are prominent after a burn. Here, we show that administration of propranolol, a nonselective β1/2 adrenergic receptor antagonist, attenuates ER stress and JNK activation. Attenuation of ER stress by propranolol results in increased insulin sensitivity, as determined by activation of hepatic phosphatidylinositol 3-kinase and Akt. We conclude that catecholamine release is responsible for the ER stress response and impaired insulin receptor signaling after burn injury. PMID:22396018

  13. Rho-Associated Kinase Inhibitors Promote Microglial Uptake Via the ERK Signaling Pathway.

    PubMed

    Fu, Peicai; Tang, Ronghua; Yu, Zhiyuan; Li, Caihong; Chen, Xue; Xie, Minjie; Wang, Wei; Luo, Xiang

    2016-02-01

    Microglia are immunocompetent cells in the central nervous system that take up tissue debris and pathogens. Rho-associated kinase (ROCK) has been identified as an important regulator of uptake, proliferation, secretion, and differentiation in a number of cell types. Although ROCK plays critical roles in the microglial secretion of inflammatory factors, migration, and morphology, its effects on microglial uptake activity have not been well characterized. In the present study, we found that treatment of BV2 microglia and primary microglia with the ROCK inhibitors Y27632 and fasudil increased uptake activity and was associated with morphological changes. Furthermore, western blots showed that this increase in uptake activity was mediated through the extracellular-signal-regulated kinase (ERK) signaling cascade, indicating the importance of ROCK in regulating microglial uptake activity. PMID:26779919

  14. Hydrogen Peroxide Sensing and Signaling by Protein Kinases in the Cardiovascular System

    PubMed Central

    Burgoyne, Joseph R.; Oka, Shin-ichi; Ale-Agha, Niloofar

    2013-01-01

    Abstract Significance: Oxidants were once principally considered perpetrators of injury and disease. However, this has become an antiquated view, with cumulative evidence showing that the oxidant hydrogen peroxide serves as a signaling molecule. Hydrogen peroxide carries vital information about the redox state of the cell and is crucial for homeostatic regulation during health and adaptation to stress. Recent Advances: In this review, we examine the contemporary concepts for how hydrogen peroxide is sensed and transduced into a biological response by introducing post-translational oxidative modifications on select proteins. Oxidant sensing and signaling by kinases are of particular importance as they integrate oxidant signals into phospho-regulated pathways. We focus on CAMKII, PKA, and PKG, kinases whose redox regulation has notable impact on cardiovascular function. Critical Issues: In addition, we examine the mechanism for regulating intracellular hydrogen peroxide, considering the net concentrations that may accumulate. The effects of endogenously generated oxidants are often modeled by applying exogenous hydrogen peroxide to cells or tissues. Here we consider whether model systems exposed to exogenous hydrogen peroxide have relevance to systems where the oxidant is generated endogenously, and if so, what concentration can be justified in terms of relevance to health and disease. Future Directions: Improving our understanding of hydrogen peroxide signaling and the sensor proteins that it can modify will help us develop new strategies to regulate intracellular signaling to prevent disease. Antioxid. Redox Signal. 18, 1042–1052. PMID:22867279

  15. Human pre-B cell receptor signal transduction: evidence for distinct roles of PI3kinase and MAP-kinase signalling pathways

    PubMed Central

    Anbazhagan, Kolandaswamy; Rabbind Singh, Amrathlal; Isabelle, Piec; Stella, Ibata; Céline, Alleaume-De Martel; Bissac, Eliane; Bertrand, Brassart; Rémy, Nyga; Naomi, Taylor; Vincent, Fuentes; Rochette, Jacques; Lassoued, Kaïss

    2013-01-01

    Pre-BCR acts as a critical checkpoint in B cell development. However, its signalling cascade still remains indistinctly characterised in human. We investigated pre-BCR signalling pathway to examine its regulation in normal primary pre-B lymphocytes and pre-B cell lines. In cell lines, early signalling events occurring after pre-BCR stimulation include phosphorylation of Lyn, Blk and Syk together with ZAP70, Btk, Vav, PLC-γ2 and various adaptor proteins, such as BLNK, LAB, LAT and SLP-76. Further downstream, these molecules induced activation of the PI3K/AKT and MAP-kinase resulting in an augmentation of canonical NF-κB pathways and cFos/AP1 activation. PI3K and MAPK exerted opposing effects on the pre-BCR-induced activation of the canonical NF-κB and c-Fos/AP1 pathways. Immediate nuclear export of FoxO3A and delayed import of IRF4 were additional events observed after pre-BCR crosslinking in primary cells. Pre-BCR-induced down-regulation of Rag1, Rag2, E2A and Pax5 transcripts occurred in a PI3K-dependent manner. Finally we bring evidence that pre-BCR stimulation or co stimulation with CD19 enhances cell cycle signal. PMID:25400915

  16. Morelloflavone, a biflavonoid, inhibits tumor angiogenesis by targeting rho GTPases and extracellular signal-regulated kinase signaling pathways.

    PubMed

    Pang, Xiufeng; Yi, Tingfang; Yi, Zhengfang; Cho, Sung Gook; Qu, Weijing; Pinkaew, Decha; Fujise, Ken; Liu, Mingyao

    2009-01-15

    Morelloflavone, a biflavonoid extracted from Garcinia dulcis, has shown antioxidative, antiviral, and anti-inflammatory properties. However, the function and the mechanism of this compound in cancer treatment and tumor angiogenesis have not been elucidated to date. In this study, we postulated that morelloflavone might have the ability to inhibit angiogenesis, the pivotal step in tumor growth, invasiveness, and metastasis. We showed that morelloflavone could inhibit vascular endothelial growth factor (VEGF)-induced cell proliferation, migration, invasion, and capillary-like tube formation of primary cultured human umbilical vascular endothelial cells in a dose-dependent manner. Morelloflavone effectively inhibited microvessel sprouting of endothelial cells in the mouse aortic ring assay and the formation of new blood microvessels induced by VEGF in the mouse Matrigel plug assay. Furthermore, morelloflavone inhibited tumor growth and tumor angiogenesis of prostate cancer cells (PC-3) in xenograft mouse tumor model in vivo, suggesting that morelloflavone inhibited tumorigenesis by targeting angiogenesis. To understand the underlying mechanism of morelloflavone on the inhibitory effect of tumor growth and angiogenesis, we showed that morelloflavone could inhibit the activation of both RhoA and Rac1 GTPases but have little effect on the activation of Cdc42 GTPase. Additionally, morelloflavone inhibited the phosphorylation and activation of Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK pathway kinases without affecting VEGF receptor 2 activity. Together, our results indicate that morelloflavone exerts antiangiogenic action by targeting the activation of Rho-GTPases and ERK signaling pathways. These findings are the first to reveal the novel functions of morelloflavone in tumor angiogenesis and its molecular basis for the anticancer action. PMID:19147565

  17. Dermatophytes Activate Skin Keratinocytes via Mitogen-Activated Protein Kinase Signaling and Induce Immune Responses

    PubMed Central

    Achterman, Rebecca R.; Moyes, David L.; Thavaraj, Selvam; Smith, Adam R.; Blair, Kris M.

    2015-01-01

    Dermatophytes cause superficial and cutaneous fungal infections in immunocompetent hosts and invasive disease in immunocompromised hosts. However, the host mechanisms that regulate innate immune responses against these fungi are largely unknown. Here, we utilized commercially available epidermal tissues and primary keratinocytes to assess (i) damage induction by anthropophilic, geophilic, and zoophilic dermatophyte strains and (ii) the keratinocyte signaling pathways, transcription factors, and proinflammatory responses induced by a representative dermatophyte, Trichophyton equinum. Initially, five dermatophyte species were tested for their ability to invade, cause tissue damage, and induce cytokines, with Microsporum gypseum inducing the greatest level of damage and cytokine release. Using T. equinum as a representative dermatophyte, we found that the mitogen-activated protein kinase (MAPK) pathways were predominantly affected, with increased levels of phospho-p38 and phospho-Jun N-terminal protein kinase (JNK) but decreased levels of phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2). Notably, the NF-κB and PI3K pathways were largely unaffected. T. equinum also significantly increased expression of the AP-1-associated transcription factor, c-Fos, and the MAPK regulatory phosphatase, MKP1. Importantly, the ability of T. equinum to invade, cause tissue damage, activate signaling and transcription factors, and induce proinflammatory responses correlated with germination, indicating that germination may be important for dermatophyte virulence and host immune activation. PMID:25667269

  18. Alternative Activation Mechanisms of Protein Kinase B Trigger Distinct Downstream Signaling Responses.

    PubMed

    Balzano, Deborah; Fawal, Mohamad-Ali; Velázquez, Jose V; Santiveri, Clara M; Yang, Joshua; Pastor, Joaquín; Campos-Olivas, Ramón; Djouder, Nabil; Lietha, Daniel

    2015-10-01

    Protein kinase B (PKB/Akt) is an important mediator of signals that control various cellular processes including cell survival, growth, proliferation, and metabolism. PKB promotes these processes by phosphorylating many cellular targets, which trigger distinct downstream signaling events. However, how PKB is able to selectively target its substrates to induce specific cellular functions remains elusive. Here we perform a systematic study to dissect mechanisms that regulate intrinsic kinase activity versus mechanisms that specifically regulate activity toward specific substrates. We demonstrate that activation loop phosphorylation and the C-terminal hydrophobic motif are essential for high PKB activity in general. On the other hand, we identify membrane targeting, which for decades has been regarded as an essential step in PKB activation, as a mechanism mainly affecting substrate selectivity. Further, we show that PKB activity in cells can be triggered independently of PI3K by initial hydrophobic motif phosphorylation, presumably through a mechanism analogous to other AGC kinases. Importantly, different modes of PKB activation result in phosphorylation of distinct downstream targets. Our data indicate that specific mechanisms have evolved for signaling nodes, like PKB, to select between various downstream events. Targeting such mechanisms selectively could facilitate the development of therapeutics that might limit toxic side effects. PMID:26286748

  19. Multiple signals modulate the activity of the complex sensor kinase TodS

    PubMed Central

    Silva-Jiménez, Hortencia; Ortega, Álvaro; García-Fontana, Cristina; Ramos, Juan Luis; Krell, Tino

    2015-01-01

    The reason for the existence of complex sensor kinases is little understood but thought to lie in the capacity to respond to multiple signals. The complex, seven-domain sensor kinase TodS controls in concert with the TodT response regulator the expression of the toluene dioxygenase pathway in Pseudomonas putida F1 and DOT-T1E. We have previously shown that some aromatic hydrocarbons stimulate TodS activity whereas others behave as antagonists. We show here that TodS responds in addition to the oxidative agent menadione. Menadione but no other oxidative agent tested inhibited TodS activity in vitro and reduced PtodX expression in vivo. The menadione signal is incorporated by a cysteine-dependent mechanism. The mutation of the sole conserved cysteine of TodS (C320) rendered the protein insensitive to menadione. We evaluated the mutual opposing effects of toluene and menadione on TodS autophosphorylation. In the presence of toluene, menadione reduced TodS activity whereas toluene did not stimulate activity in the presence of menadione. It was shown by others that menadione increases expression of glucose metabolism genes. The opposing effects of menadione on glucose and toluene metabolism may be partially responsible for the interwoven regulation of both catabolic pathways. This work provides mechanistic detail on how complex sensor kinases integrate different types of signal molecules. PMID:24986263

  20. HCMV pUS28 initiates pro-migratory signaling via activation of Pyk2 kinase

    SciTech Connect

    Vomaske, Jennifer; Varnum, Susan M.; Melnychuk, Ryan; Smith, Patricia; Pasa-Tolic, Ljiljana; Shutthanandan, Janani I.; Streblow, Daniel N.

    2010-12-10

    The HCMV-encoded chemokine receptor US28 mediates smooth muscle cell (SMC) and macrophage motility and this activity has been implicated in the acceleration of vascular disease. US28 induced SMC migration involves the activation of the protein tyrosine kinases (PTKs) Src and Focal adhesion kinase as well as the small GTPase RhoA. In the current study, we examined the involvement of the PTK Pyk2 in US28-induced cellular motility. Expression of a Pyk2 lacking the autophosphorylation site (Tyr-402) blocks US28-mediated SMC migration in response to RANTES, while the kinase-inactive mutant failed to elicit the same negative effect on migration. US28 stimulation with RANTES results in ligand-dependent and calcium-dependent phosphorylation of Pyk2 Tyr-402 and induced the formation of an active Pyk2 kinase complex containing several novel Pyk2 binding proteins. Interestingly, expression of the autophosphorylation site mutant Pyk2 F402Y did not abrogate the formation of an active Pyk2 kinase complex, but instead prevented US28-mediated activation of RhoA. These findings represent the first demonstration that US28 signals through Pyk2 and that this PTK participates in US28-mediated cellular motility via activation of RhoA. Additionally, US28 activated RhoA via Pyk2 in the U373 glioblastoma cells. Interestingly, the Pyk2 kinase complex in U373 contained several proteins known to participate in glioma tumorigenesis. These results provide a potential mechanistic link between HCMV-US28 and glioblastoma cell activation and motility.

  1. Extracellular signal-regulated kinases 1 and 2 activation in endothelial cells exposed to cyclic strain

    NASA Technical Reports Server (NTRS)

    Ikeda, M.; Takei, T.; Mills, I.; Kito, H.; Sumpio, B. E.

    1999-01-01

    The aim of this study was to determine whether extracellular signal-regulated kinases 1/2 (ERK1/ERK2) are activated and might play a role in enhanced proliferation and morphological change induced by strain. Bovine aortic endothelial cells (BAEC) were subjected to an average of 6 or 10% strain at a rate of 60 cycles/min for up to 4 h. Cyclic strain caused strain- and time-dependent phosphorylation and activation of ERK1/ERK2. Peak phosphorylation and activation of ERK1/ERK2 induced by 10% strain were at 10 min. A specific ERK1/ERK2 kinase inhibitor, PD-98059, inhibited phosphorylation and activation of ERK1/ERK2 but did not inhibit the increased cell proliferation and cell alignment induced by strain. Treatment of BAEC with 2,5-di-tert-butyl-1, 4-benzohydroquinone, to deplete inositol trisphosphate-sensitive calcium storage, and gadolinium chloride, a Ca2+ channel blocker, did not inhibit the activation of ERK1/ERK2. Strain-induced ERK1/ERK2 activation was partly inhibited by the protein kinase C inhibitor calphostin C and completely inhibited by the tyrosine kinase inhibitor genistein. These data suggest that 1) ERK1/ERK2 are not critically involved in the strain-induced cell proliferation and orientation, 2) strain-dependent activation of ERK1/ERK2 is independent of intracellular and extracellular calcium mobilization, and 3) protein kinase C activation and tyrosine kinase regulate strain-induced activation of ERK1/ERK2.

  2. Slow Inhibition and Conformation Selective Properties of Extracellular Signal-Regulated Kinase 1 and 2 Inhibitors

    PubMed Central

    Rudolph, Johannes; Xiao, Yao; Pardi, Arthur; Ahn, Natalie G.

    2016-01-01

    The mitogen-activated protein (MAP) kinase pathway is a target for anticancer therapy, validated using inhibitors of B-Raf and MAP kinase kinase (MKK) 1 and 2. Clinical outcomes show a high frequency of acquired resistance in patient tumors, involving upregulation of activity of the MAP kinase, extracellular signal-regulated kinase (ERK) 1 and 2. Thus, inhibitors for ERK1/2 are potentially important for targeted therapeutics against cancer. The structures and potencies of different ERK inhibitors have been published, but their kinetic mechanisms have not been characterized. Here we perform enzyme kinetic studies on six representative ERK inhibitors, with potencies varying from 100 pM to 20 μM. Compounds with significant biological activity (IC50 < 100 nM) that inhibit in the subnanomolar range (Vertex-11e and SCH772984) display slow-onset inhibition and represent the first inhibitors of ERK2 known to demonstrate slow dissociation rate constants (values of 0.2 and 1.1 h−1, respectively). Furthermore, we demonstrate using kinetic competition assays that Vertex-11e binds with differing affinities to ERK2 in its inactive, unphosphorylated and active, phosphorylated forms. Finally, two-dimensional heteronuclear multiple-quantum correlation nuclear magnetic resonance experiments reveal that distinct conformational states are formed in complexes of Vertex-11e with inactive and active ERK2. Importantly, two conformers interconvert in equilibrium in the active ERK2 apoenzyme, but Vertex-11e strongly shifts the equilibrium completely to one conformer. Thus, a high-affinity, slow dissociation inhibitor stabilizes different enzyme conformations depending on the activity state of ERK2 and reveals properties of conformational selection toward the active kinase. PMID:25350931

  3. Zebrafish WNK Lysine Deficient Protein Kinase 1 (wnk1) Affects Angiogenesis Associated with VEGF Signaling

    PubMed Central

    Chen, Wen-Chuan; Kou, Fong-Ji; Lu, Jeng-Wei; Wang, Horng-Dar; Huang, Chou-Long; Yuh, Chiou-Hwa

    2014-01-01

    The WNK1 (WNK lysine deficient protein kinase 1) protein is a serine/threonine protein kinase with emerging roles in cancer. WNK1 causes hypertension and hyperkalemia when overexpressed and cardiovascular defects when ablated in mice. In this study, the role of Wnk1 in angiogenesis was explored using the zebrafish model. There are two zebrafish wnk1 isoforms, wnk1a and wnk1b, and both contain all the functional domains found in the human WNK1 protein. Both isoforms are expressed in the embryo at the initiation of angiogenesis and in the posterior cardinal vein (PCV), similar to fms-related tyrosine kinase 4 (flt4). Using morpholino antisense oligonucleotides against wnk1a and wnk1b, we observed that wnk1 morphants have defects in angiogenesis in the head and trunk, similar to flk1/vegfr2 morphants. Furthermore, both wnk1a and wnk1b mRNA can partially rescue the defects in vascular formation caused by flk1/vegfr2 knockdown. Mutation of the kinase domain or the Akt/PI3K phosphorylation site within wnk1 destroys this rescue capability. The rescue experiments provide evidence that wnk1 is a downstream target for Vegfr2 (vascular endothelial growth factor receptor-2) and Akt/PI3K signaling and thereby affects angiogenesis in zebrafish embryos. Furthermore, we found that knockdown of vascular endothelial growth factor receptor-2 (flk1/vegfr2) or vascular endothelial growth factor receptor-3 (flt4/vegfr3) results in a decrease in wnk1a expression, as assessed by in situ hybridization and q-RT-PCR analysis. Thus, the Vegf/Vegfr signaling pathway controls angiogenesis in zebrafish via Akt kinase-mediated phosphorylation and activation of Wnk1 as well as transcriptional regulation of wnk1 expression. PMID:25171174

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

    PubMed Central

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

    2015-01-01

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

  5. Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases

    NASA Technical Reports Server (NTRS)

    Levonen, A. L.; Patel, R. P.; Brookes, P.; Go, Y. M.; Jo, H.; Parthasarathy, S.; Anderson, P. G.; Darley-Usmar, V. M.

    2001-01-01

    Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.

  6. Chemical Genetics Approach Reveals Importance of cAMP and MAP Kinase Signaling to Lipid and Carotenoid Biosynthesis in Microalgae.

    PubMed

    Choi, Yoon-E; Rhee, Jin-Kyu; Kim, Hyun-Soo; Ahn, Joon-Woo; Hwang, Hyemin; Yang, Ji-Won

    2015-05-01

    In this study, we attempted to understand signaling pathways behind lipid biosynthesis by employing a chemical genetics approach based on small molecule inhibitors. Specific signaling inhibitors of MAP kinase or modulators of cAMP signaling were selected to evaluate the functional roles of each of the key signaling pathways in three different microalgal species: Chlamydomonas reinhardtii, Chlorella vulgaris, and Haematococcus pluvialis. Our results clearly indicate that cAMP signaling pathways are indeed positively associated with microalgal lipid biosynthesis. In contrast, MAP kinase pathways in three microalgal species are all negatively implicated in both lipid and carotenoid biosynthesis. PMID:25563422

  7. Induction of Central Host Signaling Kinases during Pneumococcal Infection of Human THP-1 Cells.

    PubMed

    Kohler, Thomas P; Scholz, Annemarie; Kiachludis, Delia; Hammerschmidt, Sven

    2016-01-01

    Streptococcus pneumoniae is a widespread colonizer of the mucosal epithelia of the upper respiratory tract of human. However, pneumococci are also responsible for numerous local as well as severe systemic infections, especially in children under the age of five and the elderly. Under certain conditions, pneumococci are able to conquer the epithelial barrier, which can lead to a dissemination of the bacteria into underlying tissues and the bloodstream. Here, specialized macrophages represent an essential part of the innate immune system against bacterial intruders. Recognition of the bacteria through different receptors on the surface of macrophages leads thereby to an uptake and elimination of bacteria. Accompanied cytokine release triggers the migration of leukocytes from peripheral blood to the site of infection, where monocytes differentiate into mature macrophages. The rearrangement of the actin cytoskeleton during phagocytosis, resulting in the engulfment of bacteria, is thereby tightly regulated by receptor-mediated phosphorylation cascades of different protein kinases. The molecular cellular processes including the modulation of central protein kinases are only partially solved. In this study, the human monocytic THP-1 cell line was used as a model system to examine the activation of Fcγ and complement receptor-independent signal cascades during infection with S. pneumoniae. Pneumococci cultured either in chemically defined or complex medium showed no significant differences in pneumococcal phagocytosis by phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 cells. Double immuno-fluorescence microscopy and antibiotic protection assays demonstrated a time-dependent uptake and killing of S. pneumoniae 35A inside of macrophages. Infections of THP-1 cells in the presence of specific pharmacological inhibitors revealed a crucial role of actin polymerization and importance of the phosphoinositide 3-kinase (PI3K) and Protein kinase B (Akt) as well during

  8. Induction of Central Host Signaling Kinases during Pneumococcal Infection of Human THP-1 Cells

    PubMed Central

    Kohler, Thomas P.; Scholz, Annemarie; Kiachludis, Delia; Hammerschmidt, Sven

    2016-01-01

    Streptococcus pneumoniae is a widespread colonizer of the mucosal epithelia of the upper respiratory tract of human. However, pneumococci are also responsible for numerous local as well as severe systemic infections, especially in children under the age of five and the elderly. Under certain conditions, pneumococci are able to conquer the epithelial barrier, which can lead to a dissemination of the bacteria into underlying tissues and the bloodstream. Here, specialized macrophages represent an essential part of the innate immune system against bacterial intruders. Recognition of the bacteria through different receptors on the surface of macrophages leads thereby to an uptake and elimination of bacteria. Accompanied cytokine release triggers the migration of leukocytes from peripheral blood to the site of infection, where monocytes differentiate into mature macrophages. The rearrangement of the actin cytoskeleton during phagocytosis, resulting in the engulfment of bacteria, is thereby tightly regulated by receptor-mediated phosphorylation cascades of different protein kinases. The molecular cellular processes including the modulation of central protein kinases are only partially solved. In this study, the human monocytic THP-1 cell line was used as a model system to examine the activation of Fcγ and complement receptor-independent signal cascades during infection with S. pneumoniae. Pneumococci cultured either in chemically defined or complex medium showed no significant differences in pneumococcal phagocytosis by phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 cells. Double immuno-fluorescence microscopy and antibiotic protection assays demonstrated a time-dependent uptake and killing of S. pneumoniae 35A inside of macrophages. Infections of THP-1 cells in the presence of specific pharmacological inhibitors revealed a crucial role of actin polymerization and importance of the phosphoinositide 3-kinase (PI3K) and Protein kinase B (Akt) as well during

  9. p38 and Extracellular Signal-Regulated Kinases Regulate the Myogenic Program at Multiple Steps

    PubMed Central

    Wu, Zhenguo; Woodring, Pamela J.; Bhakta, Kunjan S.; Tamura, Kumiko; Wen, Fang; Feramisco, James R.; Karin, Michael; Wang, Jean Y. J.; Puri, Pier Lorenzo

    2000-01-01

    The extracellular signals which regulate the myogenic program are transduced to the nucleus by mitogen-activated protein kinases (MAPKs). We have investigated the role of two MAPKs, p38 and extracellular signal-regulated kinase (ERK), whose activities undergo significant changes during muscle differentiation. p38 is rapidly activated in myocytes induced to differentiate. This activation differs from those triggered by stress and cytokines, because it is not linked to Jun–N-terminal kinase stimulation and is maintained during the whole process of myotube formation. Moreover, p38 activation is independent of a parallel promyogenic pathway stimulated by insulin-like growth factor 1. Inhibition of p38 prevents the differentiation program in myogenic cell lines and human primary myocytes. Conversely, deliberate activation of endogenous p38 stimulates muscle differentiation even in the presence of antimyogenic cues. Much evidence indicates that p38 is an activator of MyoD: (i) p38 kinase activity is required for the expression of MyoD-responsive genes, (ii) enforced induction of p38 stimulates the transcriptional activity of a Gal4-MyoD fusion protein and allows efficient activation of chromatin-integrated reporters by MyoD, and (iii) MyoD-dependent myogenic conversion is reduced in mouse embryonic fibroblasts derived from p38α−/− embryos. Activation of p38 also enhances the transcriptional activities of myocyte enhancer binding factor 2A (MEF2A) and MEF2C by direct phosphorylation. With MEF2C, selective phosphorylation of one residue (Thr293) is a tissue-specific activating signal in differentiating myocytes. Finally, ERK shows a biphasic activation profile, with peaks of activity in undifferentiated myoblasts and postmitotic myotubes. Importantly, activation of ERK is inhibitory toward myogenic transcription in myoblasts but contributes to the activation of myogenic transcription and regulates postmitotic responses (i.e., hypertrophic growth) in myotubes. PMID

  10. AMP-Activated Protein Kinase Signalling in Cancer and Cardiac Hypertrophy

    PubMed Central

    Lipovka, Yulia; Konhilas, John P

    2015-01-01

    The AMP-protein kinase (AMPK) pathway is very versatile as it regulates cellular energetic homeostasis in many different tissue types. An appreciation for the importance of AMPK signalling and regulation in cardiovascular and tumor biology is increasing. Recently, a link has been established between anti-cancer therapy and susceptibility to cardiac disease. It has been shown that some anti-cancer drugs lead to an increased risk of cardiac disease, underlined by de-regulation of AMPK signalling. This review explores the AMPK signalling axis in both cardiac and tumor metabolism. We then examine off-target AMPK inhibition by cancer drugs and how this may translate into increased risk of cardiovascular disease. Finally, we discuss the implication of deregulated AMPK signalling during different stages of cardiac hypertrophy. Better understanding of the molecular pathways behind pathological processes will lead to the development of more effective therapeutics for cancer and cardiovascular diseases. PMID:26798768

  11. Early redox, Src family kinase, and calcium signaling integrate wound responses and tissue regeneration in zebrafish

    PubMed Central

    Yoo, Sa Kan; Freisinger, Christina M.; LeBert, Danny C.

    2012-01-01

    Tissue injury can lead to scar formation or tissue regeneration. How regenerative animals sense initial tissue injury and transform wound signals into regenerative growth is an unresolved question. Previously, we found that the Src family kinase (SFK) Lyn functions as a redox sensor in leukocytes that detects H2O2 at wounds in zebrafish larvae. In this paper, using zebrafish larval tail fins as a model, we find that wounding rapidly activated SFK and calcium signaling in epithelia. The immediate SFK and calcium signaling in epithelia was important for late epimorphic regeneration of amputated fins. Wound-induced activation of SFKs in epithelia was dependent on injury-generated H2O2. A SFK member, Fynb, was responsible for fin regeneration. This work provides a new link between early wound responses and late regeneration and suggests that redox, SFK, and calcium signaling are immediate “wound signals” that integrate early wound responses and late epimorphic regeneration. PMID:23045550

  12. Spatial and Temporal Regulation of Receptor Tyrosine Kinase Activation and Intracellular Signal Transduction.

    PubMed

    Bergeron, John J M; Di Guglielmo, Gianni M; Dahan, Sophie; Dominguez, Michel; Posner, Barry I

    2016-06-01

    Epidermal growth factor (EGF) and insulin receptor tyrosine kinases (RTKs) exemplify how receptor location is coupled to signal transduction. Extracellular binding of ligands to these RTKs triggers their concentration into vesicles that bud off from the cell surface to generate intracellular signaling endosomes. On the exposed cytosolic surface of these endosomes, RTK autophosphorylation selects the downstream signaling proteins and lipids to effect growth factor and polypeptide hormone action. This selection is followed by the recruitment of protein tyrosine phosphatases that inactivate the RTKs and deliver them by membrane fusion and fission to late endosomes. Coincidentally, proteinases inside the endosome cleave the EGF and insulin ligands. Subsequent inward budding of the endosomal membrane generates multivesicular endosomes. Fusion with lysosomes then results in RTK degradation and downregulation. Through the spatial positioning of RTKs in target cells for EGF and insulin action, the temporal extent of signaling, attenuation, and downregulation is regulated. PMID:27023845

  13. Suppression of Mitochondrial Biogenesis through Toll-Like Receptor 4–Dependent Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Signaling in Endotoxin-Induced Acute Kidney Injury

    PubMed Central

    Smith, Joshua A.; Stallons, L. Jay; Collier, Justin B.; Chavin, Kenneth D.

    2015-01-01

    Although disruption of mitochondrial homeostasis and biogenesis (MB) is a widely accepted pathophysiologic feature of sepsis-induced acute kidney injury (AKI), the molecular mechanisms responsible for this phenomenon are unknown. In this study, we examined the signaling pathways responsible for the suppression of MB in a mouse model of lipopolysaccharide (LPS)-induced AKI. Downregulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of MB, was noted at the mRNA level at 3 hours and protein level at 18 hours in the renal cortex, and was associated with loss of renal function after LPS treatment. LPS-mediated suppression of PGC-1α led to reduced expression of downstream regulators of MB and electron transport chain proteins along with a reduction in renal cortical mitochondrial DNA content. Mechanistically, Toll-like receptor 4 (TLR4) knockout mice were protected from renal injury and disruption of MB after LPS exposure. Immunoblot analysis revealed activation of tumor progression locus 2/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (TPL-2/MEK/ERK) signaling in the renal cortex by LPS. Pharmacologic inhibition of MEK/ERK signaling attenuated renal dysfunction and loss of PGC-1α, and was associated with a reduction in proinflammatory cytokine (e.g., tumor necrosis factor-α [TNF-α], interleukin-1β) expression at 3 hours after LPS exposure. Neutralization of TNF-α also blocked PGC-1α suppression, but not renal dysfunction, after LPS-induced AKI. Finally, systemic administration of recombinant tumor necrosis factor-α alone was sufficient to produce AKI and disrupt mitochondrial homeostasis. These findings indicate an important role for the TLR4/MEK/ERK pathway in both LPS-induced renal dysfunction and suppression of MB. TLR4/MEK/ERK/TNF-α signaling may represent a novel therapeutic target to prevent mitochondrial dysfunction and AKI produced by sepsis. PMID:25503387

  14. Role of Fyn kinase in signaling associated with epiboly during zebrafish development.

    PubMed

    Sharma, Dipika; Holets, Lesya; Zhang, Xiaoming; Kinsey, William H

    2005-09-15

    The function of Fyn kinase during zebrafish development through the blastula stage was investigated through the use of dominant-negative constructs designed to suppress the function of zebrafish c-Fyn. Microinjection of SH2 domain-containing fusion protein or mRNA encoding the mutated, catalytically inactive Fyn at 45 min post-insemination had no significant effect during cleavage and did not inhibit formation of the yolk syncitial layer. Smoothing of the enveloping cell layer at the midblastula transition occurred normally and expression of bon/mixer and mezzo, zygotic transcription factors indicated that activation of the zygotic genome did occur. Signaling pathways involved with axis determination such as beta-catenin, activin, and nodal appeared to function normally as evidenced by expression of boz, goosecoid, and mezzo. However, while formation of the yolk syncitial layer was normal, the marginal blastomeres failed to migrate toward the vegetal pole and epiboly did not occur, a phenotype similar but distinct from that resulting from suppression of c-Yes kinase. The block to development was prevented by co-injection of c-Fyn mRNA with the dominant-negative construct indicating that it was a specific effect. Injection of the dominant-negative mRNA into individual blastomeres indicated that the effect was exerted on the intrinsic ability of the individual blastomeres to respond to signals directing epiboly and not on the signals themselves. Analysis of the pattern of calcium signaling in experimental and control embryos demonstrated that the elevated [Ca2+]i characteristic of the marginal blastomeres was suppressed. Together, these observations indicate that Fyn kinase plays an important role in epiboly, possibly through its effects in calcium signaling. PMID:16112104

  15. Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants

    PubMed Central

    Klein, Pontus; Müller-Rischart, Anne Kathrin; Motori, Elisa; Schönbauer, Cornelia; Schnorrer, Frank; Winklhofer, Konstanze F; Klein, Rüdiger

    2014-01-01

    Parkinson's disease (PD)-associated Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking. Glial cell line-derived neurotrophic factor (GDNF) and its signaling receptor Ret are neuroprotective in toxin-based animal models of PD. However, the mechanism by which GDNF/Ret protects cells from degenerating remains unclear. We investigated whether the Drosophila homolog of Ret can rescue Pink1 and park mutant phenotypes. We report that a signaling active version of Ret (RetMEN2B) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants. Interestingly, corresponding phenotypes of park mutants were not rescued, suggesting that the phenotypes of Pink1 and park mutants have partially different origins. In human neuroblastoma cells, GDNF treatment rescues morphological defects of PINK1 knockdown, without inducing mitophagy or Parkin recruitment. GDNF also rescues bioenergetic deficits of PINK knockdown cells. Furthermore, overexpression of RetMEN2B significantly improves electron transport chain complex I function in Pink1 mutant Drosophila. These results provide a novel mechanism underlying Ret-mediated cell protection in a situation relevant for human PD. PMID:24473149

  16. Apelin/APJ signaling promotes hypoxia-induced proliferation of endothelial progenitor cells via phosphoinositide-3 kinase/Akt signaling.

    PubMed

    Zhang, Jingchang; Liu, Qiming; Hu, Xinqun; Fang, Zhenfei; Huang, Feng; Tang, Liang; Zhou, Shenghua

    2015-09-01

    Endothelial progenitor cells (EPCs) can adhere to the endothelium at sites of hypoxia/ischemia and participate in the formation of novel vessels through differentiating into endothelial cells (ECs). Apelin is an endogenous ligand for the G protein‑coupled receptor APJ, and apelin/APJ signaling has a role in cardiovascular function. The present study aimed to investigate the role of apelin/APJ signaling in the regulation of EPC proliferation under hypoxia. The results showed that hypoxia was able to induce EPC proliferation, accompanied with an upregulation of hypoxia‑inducible factor (HIF)‑1α as well as apelin/APJ signaling. Further investigation indicated that siRNA‑mediated knockdown of apelin or APJ expression attenuated the hypoxia‑induced proliferation of EPCs, suggesting that apelin/APJ signaling has an important role in hypoxia‑induced EPC proliferation. Moreover, the phosphoinositide‑3 kinase (PI3K)/Akt signaling pathway was found to be involved in the apelin/APJ‑mediated EPC proliferation under hypoxia. Based on these findings, the present study suggested that hypoxia‑induced upregulation of HIF‑1α promotes the expression of apelin and APJ, which further activate the downstream PI3K/Akt signaling pathway, a key promoter of EPC proliferation. In conclusion, the present study highlighted the role of apelin/APJ in the regulation of EPC proliferation, and apelin/APJ may therefore serve as a potential target for the prevention of hypoxic ischemic injury. PMID:26018184

  17. Protein Kinase C alpha (PKCα) dependent signaling mediates endometrial cancer cell growth and tumorigenesis

    PubMed Central

    Haughian, James M.; Reno, Elaine M.; Thorne, Alicia M.; Bradford, Andrew P.

    2009-01-01

    Endometrial cancer is the most common invasive gynecologic malignancy, yet molecular mechanisms and signaling pathways underlying its etiology and pathophysiology remain poorly characterized. We sought to define a functional role for the protein kinase C (PKC) isoform, PKCα, in an established cell model of endometrial adenocarcinoma. Ishikawa cells depleted of PKCα protein grew slower, formed fewer colonies in anchorage-independent growth assays and exhibited impaired xenograft tumor formation in nude mice. Consistent with impaired growth, PKCα knockdown increased levels of the cyclin dependent kinase (CDK) inhibitors p21Cip1/WAF1 (p21) and p27Kip1 (p27). Despite the absence of functional phosphatase and tensin homologue (PTEN) protein in Ishikawa cells, PKCα knockdown reduced Akt phosphorylation at serine 473 and concomitantly inhibited phosphorylation of the Akt target, glycogen synthase kinase-3β (GSK-3β). PKCα knockdown also resulted in decreased basal ERK phosphorylation and attenuated ERK activation following EGF stimulation. p21 and p27 expression was not increased by treatment of Ishikawa cells with ERK and Akt inhibitors, suggesting PKCα regulates CDK expression independently of Akt and ERK. Immunohistochemical analysis of grade 1 endometrioid adenocarcinoma revealed aberrant PKCα expression, with foci of elevated PKCα staining, not observed in normal endometrium. These studies demonstrate a critical role for PKCα signaling in endometrial tumorigenesis by regulating expression of CDK inhibitors p21 and p27 and activation of Akt and ERK dependent proliferative pathways. Thus, targeting PKCα may provide novel therapeutic options in endometrial tumors. PMID:19672862

  18. Activation of JAK2 kinase mediated by the interleukin 6 signal transducer gp130.

    PubMed Central

    Narazaki, M; Witthuhn, B A; Yoshida, K; Silvennoinen, O; Yasukawa, K; Ihle, J N; Kishimoto, T; Taga, T

    1994-01-01

    The interleukin 6 receptor-associated signal transducer, gp130, is shared by receptor complexes for leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and interleukin 11. We show here that JAK2 kinase is rapidly tyrosine phosphorylated in mouse embryonic stem cells whose pluripotentiality is maintained only by gp130-sharing cytokines after stimulation that is known to induce gp130 homodimerization. JAK1 is also tyrosine phosphorylated, but to a lesser extent, under the same conditions. Comparable results are obtained with hemopoietic lineage cells such as myeloid leukemic M1 cells and pro-B-cell line-derived transfectants expressing gp130, the former of which differentiate into macrophages after stimulation of gp130 and the latter of which initiate DNA synthesis. gp130-dimerizing stimulus upregulates kinase activity of JAK2 as revealed by immunocomplex kinase assay. Deletion or point mutation in the membrane-proximal cytoplasmic motifs in gp130 that are conserved in the hemopoietic cytokine receptor family results in the loss of tyrosine phosphorylation of JAK2, which coincides with the lack of signal transducing capability of gp130 mutants. Images PMID:8134389

  19. Irisin promotes osteoblast proliferation and differentiation via activating the MAP kinase signaling pathways.

    PubMed

    Qiao, Xiaoyong; Yong Qiao, Xiao; Nie, Ying; Ma, Yaxian; Xian Ma, Ya; Chen, Yan; Cheng, Ran; Yin, Weiyao; Yao Yinrg, Wei; Hu, Ying; Xu, Wenming; Ming Xu, Wen; Xu, Liangzhi; Zhi Xu, Liang

    2016-01-01

    Physical exercise is able to improve skeletal health. However, the mechanisms are poorly known. Irisin, a novel exercise-induced myokine, secreted by skeletal muscle in response to exercise, have been shown to mediate beneficial effects of exercise in many disorders. In the current study, we demonstrated that irisin promotes osteoblast proliferation, and increases the expression of osteoblastic transcription regulators, such as Runt-related transcription factor-2, osterix/sp7; and osteoblast differentiation markers, including alkaline phosphatase, collagen type 1 alpha-1, osteocalcin, and osteopontin in vitro. Irisin also increase ALP activity and calcium deposition in cultured osteoblast. These osteogenic effects were mediated by activating the p38 mitogen-activated protein kinase (p-p38 MAPK) and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK by SB023580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on Runx2 expression and ALP activity. Together our observation suggest that irisin directly targets osteoblast, promoting osteoblast proliferation and differentiation via activating P38/ERK MAP kinase signaling cascades in vitro. Whether irisin can be utilized as the therapeutic agents for osteopenia and osteoporosis is worth to be further pursued. PMID:26738434

  20. Janus Kinase 1 Is Essential for Inflammatory Cytokine Signaling and Mammary Gland Remodeling.

    PubMed

    Sakamoto, Kazuhito; Wehde, Barbara L; Yoo, Kyung Hyun; Kim, Taemook; Rajbhandari, Nirakar; Shin, Ha Youn; Triplett, Aleata A; Rädler, Patrick D; Schuler, Fabian; Villunger, Andreas; Kang, Keunsoo; Hennighausen, Lothar; Wagner, Kay-Uwe

    2016-06-01

    Despite a wealth of knowledge about the significance of individual signal transducers and activators of transcription (STATs), essential functions of their upstream Janus kinases (JAKs) during postnatal development are less well defined. Using a novel mammary gland-specific JAK1 knockout model, we demonstrate here that this tyrosine kinase is essential for the activation of STAT1, STAT3, and STAT6 in the mammary epithelium. The loss of JAK1 uncouples interleukin-6-class ligands from their downstream effector, STAT3, which leads to the decreased expression of STAT3 target genes that are associated with the acute-phase response, inflammation, and wound healing. Consequently, JAK1-deficient mice exhibit impaired apoptosis and a significant delay in mammary gland remodeling. Using RNA sequencing, we identified several new JAK1 target genes that are upregulated during involution. These include Bmf and Bim, which are known regulators of programmed cell death. Using a BMF/BIM-double-knockout epithelial transplant model, we further validated that the synergistic action of these proapoptotic JAK1 targets is obligatory for the remodeling of the mammary epithelium. The collective results of this study suggest that JAK1 has nonredundant roles in the activation of particular STAT proteins and this tyrosine kinase is essential for coupling inflammatory cytokine signals to the cell death machinery in the differentiated mammary epithelium. PMID:27044867

  1. G12 Signaling through c-Jun NH2-Terminal Kinase Promotes Breast Cancer Cell Invasion

    PubMed Central

    Juneja, Juhi; Cushman, Ian; Casey, Patrick J.

    2011-01-01

    Signaling through the heterotrimeric G protein, G12, via Rho induces a striking increase in breast cancer cell invasion. In this study, evidence is provided that the c-Jun NH2-terminal kinase (JNK) is a key downstream effector of G12 on this pathway. Expression of constitutively-active Gα12 or activation of G12 signaling by thrombin leads to increased JNK and c-Jun phosphorylation. Pharmacologic inhibition of JNK or knockdown of JNK expression by siRNA significantly decreases G12-induced JNK activation as well as the ability of breast cancer cells to invade a reconstituted basement membrane. Furthermore, expression of dominant-negative Rho or treatment of cells with an inhibitor of the Rho kinase, ROCK, reduces G12-induced JNK and c-Jun activation, and ROCK inhibitor treatment also inhibits G12-induced cellular invasion. JNK knockdown or ROCK inhibitor treatment has no effect on activation of Rho by G12. Taken together, our data indicate that JNK activation is required for G12-induced invasion of breast cancer cells and that JNK is downstream of Rho and ROCK on this pathway. This study implicates a G12-stimulated mitogen-activated protein kinase cascade in cancer cell invasion, and supports a role for JNK in cancer progression. PMID:22087220

  2. Spatial Organization in Protein Kinase A Signaling Emerged at the Base of Animal Evolution.

    PubMed

    Peng, Mao; Aye, Thin Thin; Snel, Berend; van Breukelen, Bas; Scholten, Arjen; Heck, Albert J R

    2015-07-01

    In phosphorylation-directed signaling, spatial and temporal control is organized by complex interaction networks that diligently direct kinases toward distinct substrates to fine-tune specificity. How these protein networks originate and evolve into complex regulatory machineries are among the most fascinating research questions in biology. Here, spatiotemporal signaling is investigated by tracing the evolutionary dynamics of each functional domain of cAMP-dependent protein kinase (PKA) and its diverse set of A-kinase anchoring proteins (AKAPs). Homologues of the catalytic (PKA-C) and regulatory (PKA-R) domains of the (PKA-R)2-(PKA-C)2 holoenzyme were found throughout evolution. Most variation was observed in the RIIa of PKA-R, crucial for dimerization and docking to AKAPs. The RIIa domain was not observed in all PKA-R homologues. In the fungi and distinct protist lineages, the RIIa domain emerges within PKA-R, but it displays large sequence variation. These organisms do not harbor homologues of AKAPs, suggesting that efficient docking to direct spatiotemporal PKA activity evolved in multicellular eukaryotes. To test this in silico hypothesis, we experimentally screened organisms with increasing complexity by cAMP-based chemical proteomics to reveal that the occurrence of PKA-AKAP interactions indeed coincided and expanded within vertebrates, suggesting a crucial role for AKAPs in the advent of metazoan multicellularity. PMID:26066639

  3. Irisin promotes osteoblast proliferation and differentiation via activating the MAP kinase signaling pathways

    PubMed Central

    Yong Qiao, Xiao; Nie, Ying; Xian Ma, Ya; Chen, Yan; Cheng, Ran; Yao Yinrg, Wei; Hu, Ying; Ming Xu, Wen; Zhi Xu, Liang

    2016-01-01

    Physical exercise is able to improve skeletal health. However, the mechanisms are poorly known. Irisin, a novel exercise-induced myokine, secreted by skeletal muscle in response to exercise, have been shown to mediate beneficial effects of exercise in many disorders. In the current study, we demonstrated that irisin promotes osteoblast proliferation, and increases the expression of osteoblastic transcription regulators, such as Runt-related transcription factor-2, osterix/sp7; and osteoblast differentiation markers, including alkaline phosphatase, collagen type 1 alpha-1, osteocalcin, and osteopontin in vitro. Irisin also increase ALP activity and calcium deposition in cultured osteoblast. These osteogenic effects were mediated by activating the p38 mitogen-activated protein kinase (p-p38 MAPK) and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK by SB023580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on Runx2 expression and ALP activity. Together our observation suggest that irisin directly targets osteoblast, promoting osteoblast proliferation and differentiation via activating P38/ERK MAP kinase signaling cascades in vitro. Whether irisin can be utilized as the therapeutic agents for osteopenia and osteoporosis is worth to be further pursued. PMID:26738434

  4. Differential regulation and role of Interleukin-1 Receptor Associated Kinase-M in innate immunity signaling

    PubMed Central

    Su, Jianmin; Xie, Qifa; Wilson, Ingred; Li, Liwu

    2007-01-01

    Toll-like-receptor mediated signaling is finely regulated by a complex intracellular protein network including the interleukin-1 receptor associate kinases (IRAKs). IRAK-4, 1, and 2 may positively regulate innate immunity signaling through the activation of various downstream kinases such as MAPKs. In contrast, IRAK-M plays an inhibitory role through unknown mechanism. In this report, we show that IRAK-M is ubiquitously present in the cell, and becomes exclusively cytoplasmic upon bacterial lipoprotein Pam3CSK4 challenge. Furthermore, using bone marrow derived macrophages (BMDM) from wild type, IRAK1−/−, and IRAK-M−/− mice, we have herein demonstrated that IRAK-M selectively attenuates bacterial lipopeptide Pam3CSK4-induced p38 activation, but not ERK or JNK. IRAK1−/− and IRAK-M−/− BMDM display distinct activation profile of various MAP kinases upon Pam3CSK4 challenge, indicating that IRAK-M exerts its inhibitory effect through an IRAK1 independent pathway. Pam3CSK4 challenge leads to rapid decrease of MKP-1 protein level in IRAK-M−/− BMDM as well as THP-1 cells with decreased IRAK-M expression through siRNA interference. Our findings indicate that IRAK-M selectively attenuates p38 activation and inhibits innate immunity through stabilizing MKP-1. PMID:17379480

  5. Decoding the oxidative stress hypothesis in diabetic embryopathy through proapoptotic kinase signaling.

    PubMed

    Yang, Peixin; Reece, E Albert; Wang, Fang; Gabbay-Benziv, Rinat

    2015-05-01

    Maternal diabetes-induced birth defects occur in 6-10% of babies born to mothers with pregestational diabetes, representing a significant maternal-fetal health problem. Currently, these congenital malformations represent a significant maternal-fetal medicine issue, but are likely to create an even greater public health threat as 3 million women of reproductive age (19-44 years) have diabetes in the United States alone, and this number is expected to double by 2030. Neural tube defects (NTDs) and congenital heart defects are the most common types of birth defects associated with maternal diabetes. Animal studies have revealed that embryos under hyperglycemic conditions exhibit high levels of oxidative stress resulting from enhanced production of reactive oxygen species and impaired antioxidant capability. Oxidative stress activates a set of proapoptotic kinase signaling intermediates leading to abnormal cell death in the embryonic neural tube, which causes NTD formation. Work in animal models also has revealed that maternal diabetes triggers a series of signaling intermediates: protein kinase C (PKC) isoforms, PKCα, βII and δ; apoptosis signal-regulating kinase 1; c-Jun-N-terminal kinase (JNK)1/2; caspase; and apoptosis. Specifically, maternal diabetes in rodent models activates the proapoptotic unfolded protein response and endoplasmic reticulum (ER) stress. A reciprocal causation between JNK1/2 activation and ER stress exists in diabetic embryopathy. Molecular studies further demonstrate that deletion of the genes for Prkc, Ask1, Jnk1, or Jnk2 abolishes maternal diabetes-induced neural progenitor apoptosis and ameliorates NTD formation. Similar preventive effects are also observed when apoptosis signal-regulating kinase 1, JNK1/2, or ER stress is inhibited. Cell membrane stabilizers and antioxidant supplements are also effective in prevention of diabetes-induced birth defects. Mechanistic studies have revealed important insights into our understanding the cause

  6. Inhibition of diacylglycerol kinases as a physiological way to promote diacylglycerol signaling.

    PubMed

    Baldanzi, Gianluca

    2014-05-01

    Diacylglycerol is a key regulator of cell physiology, controlling the membrane recruitment and activation of signaling molecules. Accordingly, diacylglycerol generation and metabolism are strictly controlled, allowing for localized regulation of its concentration. While the increased production of diacylglycerol upon receptor triggering is well recognized, the modulation of diacylglycerol metabolism by diacylglycerol kinases (DGKs) is less characterized. Some agonists induce DGK activation and recruitment to the plasma membrane, promoting diacylglycerol metabolism to phosphatidic acid. Conversely, several reports indicate that signaling pathways that selectively inhibits DGK isoforms can enhance cellular diacylglycerol levels and signal transduction. For example, the impairment of DGKθ activity by RhoA binding to the catalytic domain represents a conserved mechanism controlling diacylglycerol signaling from Caenorhabditis elegans motoneurons to mammalian hepatocytes. Similarly, DGKα activity is inhibited in lymphocytes by TCR signaling, thus contributing to a rise in diacylglycerol concentration for downstream signaling. Finally, DGKμ activity is inhibited by ischemia-reperfusion-generated reactive oxygen species in airway endothelial cells, promoting diacylglycerol-mediated ion channel opening and edema. In those systems, DGKs provide a gatekeeper function by blunting diacylglycerol levels or possibly establishing permissive domains for diacylglycerol signaling. In this review, I discuss the possible general relevance of DGK inhibition to enhanced diacylglycerol signaling. PMID:24582387

  7. Receptor Tyrosine Kinase Signaling: Regulating Neural Crest Development One Phosphate at a Time

    PubMed Central

    Fantauzzo, Katherine A.; Soriano, Philippe

    2015-01-01

    Receptor tyrosine kinases (RTKs) bind to a subset of growth factors on the surface of cells and elicit responses with broad roles in developmental and postnatal cellular processes. Receptors in this subclass consist of an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular domain harboring a catalytic tyrosine kinase and regulatory sequences that are phosphorylated either by the receptor itself or various interacting proteins. Once activated, RTKs bind signaling molecules and recruit effector proteins to mediate downstream cellular responses through various intracellular signaling pathways. In this chapter, we will highlight the role of a subset of RTK families in regulating the activity of neural crest cells (NCCs) and the development of their derivatives in mammalian systems. NCCs are migratory, multipotent cells that can be subdivided into four axial populations, cranial, cardiac, vagal and trunk. These cells migrate throughout the vertebrate embryo along defined pathways and give rise to unique cell types and structures. Interestingly, individual RTK families often have specific functions in a subpopulation of NCCs that contribute to the diversity of these cells and their derivatives in the mammalian embryo. We will additionally discuss current methods used to investigate RTK signaling, including genetic, biochemical, large-scale proteomic and biosensor approaches, which can be applied to study intracellular signaling pathways active downstream of this receptor subclass during NCC development. PMID:25662260

  8. Redundant canonical and noncanonical Caenorhabditis elegans p21-activated kinase signaling governs distal tip cell migrations.

    PubMed

    Peters, Eldon C; Gossett, Andrea J; Goldstein, Bob; Der, Channing J; Reiner, David J

    2013-02-01

    p21-activated kinases (Paks) are prominent mediators of Rac/Cdc42-dependent and -independent signaling and regulate signal transduction and cytoskeletal-based cell movements. We used the reproducible migrations of the Caenorhabditis elegans gonadal distal tip cells to show that two of the three nematode Pak proteins, MAX-2 and PAK-1, function redundantly in regulation of cell migration but are regulated by very different mechanisms. First, we suggest that MAX-2 requires CED-10/Rac function and thus functions canonically. Second, PIX-1 and GIT-1 function in the same role as PAK-1, and PAK-1 interaction with PIX-1 is required for PAK-1 activity; thus, PAK-1 functions noncanonically. The human Pak-Pix-Git complex is central to noncanonical Pak signaling and requires only modest Rac/CDC-42 input. Unlike the human complex, our results suggest that the C. elegans Pak-Pix-Git complex requires PAK-1 kinase domain activity. This study delineates signaling network relationships in this cell migration model, thus providing potential further mechanistic insights and an assessment of total Pak contribution to cell migration events. PMID:23390595

  9. Potent and Selective CK2 Kinase Inhibitors with Effects on Wnt Pathway Signaling in Vivo.

    PubMed

    Dowling, James E; Alimzhanov, Marat; Bao, Larry; Chuaqui, Claudio; Denz, Christopher R; Jenkins, Emma; Larsen, Nicholas A; Lyne, Paul D; Pontz, Timothy; Ye, Qing; Holdgate, Geoff A; Snow, Lindsay; O'Connell, Nichole; Ferguson, Andrew D

    2016-03-10

    The Wnt pathway is an evolutionarily conserved and tightly regulated signaling network with important roles in embryonic development and adult tissue regeneration. Impaired Wnt pathway regulation, arising from mutations in Wnt signaling components, such as Axin, APC, and β-catenin, results in uncontrolled cell growth and triggers oncogenesis. To explore the reported link between CK2 kinase activity and Wnt pathway signaling, we sought to identify a potent, selective inhibitor of CK2 suitable for proof of concept studies in vivo. Starting from a pyrazolo[1,5-a]pyrimidine lead (2), we identified compound 7h, a potent CK2 inhibitor with picomolar affinity that is highly selectivity against other kinase family enzymes and inhibits Wnt pathway signaling (IC50 = 50 nM) in DLD-1 cells. In addition, compound 7h has physicochemical properties that are suitable for formulation as an intravenous solution, has demonstrated good pharmacokinetics in preclinical species, and exhibits a high level of activity as a monotherapy in HCT-116 and SW-620 xenografts. PMID:26985319

  10. The Tec Kinase-Regulated Phosphoproteome Reveals a Mechanism for the Regulation of Inhibitory Signals in Murine Macrophages.

    PubMed

    Tampella, Giacomo; Kerns, Hannah M; Niu, Deqiang; Singh, Swati; Khim, Socheath; Bosch, Katherine A; Garrett, Meghan E; Moguche, Albanus; Evans, Erica; Browning, Beth; Jahan, Tahmina A; Nacht, Mariana; Wolf-Yadlin, Alejandro; Plebani, Alessandro; Hamerman, Jessica A; Rawlings, David J; James, Richard G

    2015-07-01

    Previous work has shown conflicting roles for Tec family kinases in regulation of TLR-dependent signaling in myeloid cells. In the present study, we performed a detailed investigation of the role of the Tec kinases Btk and Tec kinases in regulating TLR signaling in several types of primary murine macrophages. We demonstrate that primary resident peritoneal macrophages deficient for Btk and Tec secrete less proinflammatory cytokines in response to TLR stimulation than do wild-type cells. In contrast, we found that bone marrow-derived and thioglycollate-elicited peritoneal macrophages deficient for Btk and Tec secrete more proinflammatory cytokines than do wild-type cells. We then compared the phosphoproteome regulated by Tec kinases and LPS in primary peritoneal and bone marrow-derived macrophages. From this analysis we determined that Tec kinases regulate different signaling programs in these cell types. In additional studies using bone marrow-derived macrophages, we found that Tec and Btk promote phosphorylation events necessary for immunoreceptor-mediated inhibition of TLR signaling. Taken together, our results are consistent with a model where Tec kinases (Btk, Tec, Bmx) are required for TLR-dependent signaling in many types of myeloid cells. However, our data also support a cell type-specific TLR inhibitory role for Btk and Tec that is mediated by immunoreceptor activation and signaling via PI3K. PMID:26026062