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Sample records for kinase signaling antagonizes

  1. Drosophila Tribbles Antagonizes Insulin Signaling-Mediated Growth and Metabolism via Interactions with Akt Kinase

    PubMed Central

    Das, Rahul; Sebo, Zachary; Pence, Laramie; Dobens, Leonard L.

    2014-01-01

    Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation. PMID:25329475

  2. Guanylyl cyclase/natriuretic peptide receptor-A signaling antagonizes phosphoinositide hydrolysis, Ca2+ release, and activation of protein kinase C

    PubMed Central

    Pandey, Kailash N.

    2014-01-01

    Thus far, three related natriuretic peptides (NPs) and three distinct sub-types of cognate NP receptors have been identified and characterized based on the specific ligand binding affinities, guanylyl cyclase activity, and generation of intracellular cGMP. Atrial and brain natriuretic peptides (ANP and BNP) specifically bind and activate guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), and C-type natriuretic peptide (CNP) shows specificity to activate guanylyl cyclase/natriuretic peptide receptor-B (GC-B/NPRB). All three NPs bind to natriuretic peptide receptor-C (NPRC), which is also known as clearance or silent receptor. The NPRA is considered the principal biologically active receptor of NP family; however, the molecular signaling mechanisms of NP receptors are not well understood. The activation of NPRA and NPRB produces the intracellular second messenger cGMP, which serves as the major signaling molecule of all three NPs. The activation of NPRB in response to CNP also produces the intracellular cGMP; however, at lower magnitude than that of NPRA, which is activated by ANP and BNP. In addition to enhanced accumulation of intracellular cGMP in response to all three NPs, the levels of cAMP, Ca2+ and inositol triphosphate (IP3) have also been reported to be altered in different cells and tissue types. Interestingly, ANP has been found to lower the concentrations of cAMP, Ca2+, and IP3; however, NPRC has been proposed to increase the levels of these metabolic signaling molecules. The mechanistic studies of decreased and/or increased levels of cAMP, Ca2+, and IP3 in response to NPs and their receptors have not yet been clearly established. This review focuses on the signaling mechanisms of ANP/NPRA and their biological effects involving an increased level of intracellular accumulation of cGMP and a decreased level of cAMP, Ca2+, and IP3 in different cells and tissue systems. PMID:25202235

  3. Bimodal antagonism of PKA signalling by ARHGAP36

    PubMed Central

    Eccles, Rebecca L.; Czajkowski, Maciej T.; Barth, Carolin; Müller, Paul Markus; McShane, Erik; Grunwald, Stephan; Beaudette, Patrick; Mecklenburg, Nora; Volkmer, Rudolf; Zühlke, Kerstin; Dittmar, Gunnar; Selbach, Matthias; Hammes, Annette; Daumke, Oliver; Klussmann, Enno; Urbé, Sylvie; Rocks, Oliver

    2016-01-01

    Protein kinase A is a key mediator of cAMP signalling downstream of G-protein-coupled receptors, a signalling pathway conserved in all eukaryotes. cAMP binding to the regulatory subunits (PKAR) relieves their inhibition of the catalytic subunits (PKAC). Here we report that ARHGAP36 combines two distinct inhibitory mechanisms to antagonise PKA signalling. First, it blocks PKAC activity via a pseudosubstrate motif, akin to the mechanism employed by the protein kinase inhibitor proteins. Second, it targets PKAC for rapid ubiquitin-mediated lysosomal degradation, a pathway usually reserved for transmembrane receptors. ARHGAP36 thus dampens the sensitivity of cells to cAMP. We show that PKA inhibition by ARHGAP36 promotes derepression of the Hedgehog signalling pathway, thereby providing a simple rationale for the upregulation of ARHGAP36 in medulloblastoma. Our work reveals a new layer of PKA regulation that may play an important role in development and disease. PMID:27713425

  4. RAB-7 antagonizes LET-23 EGFR signaling during vulva development in Caenorhabditis elegans.

    PubMed

    Skorobogata, Olga; Rocheleau, Christian E

    2012-01-01

    The Rab7 GTPase regulates late endosome trafficking of the Epidermal Growth Factor Receptor (EGFR) to the lysosome for degradation. However, less is known about how Rab7 activity, functioning late in the endocytic pathway, affects EGFR signaling. Here we used Caenorhabditis elegans vulva cell fate induction, a paradigm for genetic analysis of EGFR/Receptor Tyrosine Kinase (RTK) signaling, to assess the genetic requirements for rab-7. Using a rab-7 deletion mutant, we demonstrate that rab-7 antagonizes LET-23 EGFR signaling to a similar extent, but in a distinct manner, as previously described negative regulators such as sli-1 c-Cbl. Epistasis analysis places rab-7 upstream of or in parallel to lin-3 EGF and let-23 EGFR. However, expression of gfp::rab-7 in the Vulva Presursor Cells (VPCs) is sufficient to rescue the rab-7(-) VPC induction phenotypes indicating that RAB-7 functions in the signal receiving cell. We show that components of the Endosomal Sorting Complex Required for Transport (ESCRT)-0, and -I, complexes, hgrs-1 Hrs, and vps-28, also antagonize signaling, suggesting that LET-23 EGFR likely transits through Multivesicular Bodies (MVBs) en route to the lysosome. Consistent with RAB-7 regulating LET-23 EGFR trafficking, rab-7 mutants have increased number of LET-23::GFP-positive endosomes. Our data imply that Rab7, by mediating EGFR trafficking and degradation, plays an important role in downregulation of EGFR signaling. Failure to downregulate EGFR signaling contributes to oncogenesis, and thus Rab7 could possess tumor suppressor activity in humans.

  5. AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

    PubMed Central

    Johanns, M.; Lai, Y.-C.; Hsu, M.-F.; Jacobs, R.; Vertommen, D.; Van Sande, J.; Dumont, J. E.; Woods, A.; Carling, D.; Hue, L.; Viollet, B.; Foretz, M; Rider, M H

    2016-01-01

    Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation. PMID:26952277

  6. Essential role of protein kinase R antagonism by TRS1 in human cytomegalovirus replication.

    PubMed

    Braggin, Jacquelyn E; Child, Stephanie J; Geballe, Adam P

    2016-02-01

    Human cytomegalovirus (HCMV) lacking TRS1 and IRS1 (HCMV[ΔI/ΔT]) cannot replicate in cell culture. Although both proteins can block the protein kinase R (PKR) pathway, they have multiple other activities and binding partners. It remains unknown which functions are essential for HCMV replication. To investigate this issue, we first identified a TRS1 mutant that is unable to bind to PKR. Like HCMV[ΔI/ΔT], a recombinant HCMV containing this mutant (HCMV[TRS1-Mut 1]) did not replicate in wild-type cells. However, HCMV[ΔI/ΔT] did replicate in cells in which PKR expression was reduced by RNA interference. Moreover, HCMV[ΔI/ΔT] and HCMV[TRS1-Mut 1] replicated to similar levels as virus containing wild-type TRS1 in cell lines in which PKR expression was knocked out by CRISPR/Cas9-mediated genome editing. These results demonstrate that the sole essential function of TRS1 is to antagonize PKR and that its other activities do not substantially enhance HCMV replication, at least in cultured human fibroblasts.

  7. Cardiomyocyte apoptosis triggered by RAFTK/pyk2 via Src kinase is antagonized by paxillin.

    PubMed

    Melendez, Jaime; Turner, Christopher; Avraham, Hava; Steinberg, Susan F; Schaefer, Erik; Sussman, Mark A

    2004-12-17

    Altered cellular adhesion and apoptotic signaling in cardiac remodeling requires coordinated regulation of multiple constituent proteins that comprise cytoskeletal focal adhesions. One such protein activated by cardiac remodeling is related adhesion focal tyrosine kinase (RAFTK, also known as pyk2). Adenoviral-mediated expression of RAFTK in neonatal rat cardiomyocytes involves concurrent increases in phosphorylation of Src, c-Jun N-terminal kinase, and p38 leading to characteristic apoptotic changes including cleavage of poly(ADP-ribose) polymerase, caspase-3 activation, and increased DNA laddering. DNA laddering was decreased by mutation of the Tyr(402) Src-binding site in RAFTK, suggesting a central role for Src activity in apoptotic cell death that was confirmed by adenoviral-mediated Src expression. Multiple apoptotic signaling cascades are recruited by RAFTK as demonstrated by prevention of apoptosis using caspase-3 inhibitor IV (caspase-3 specific inhibitor), PP2 (Src-specific kinase inhibitor), or Csk (cellular negative regulator for Src), as well as dominant negative constructs for p38beta or MKP-1. These RAFTK-mediated phenotypic characteristics are prevented by concurrent expression of wild-type or a phosphorylation-deficient paxillin mutated at Tyr(31) and Tyr(118). Wild-type or mutant paxillin protein accumulation in the cytoplasm has no overt effect upon cell structure, but paxillin accumulation prevents losses of myofibril organization as well as focal adhesion kinase, vinculin, and paxillin protein levels mediated by RAFTK. Apoptotic signaling cascade inhibition by paxillin indicates interruption of signaling proximal to but downstream of RAFTK activity. Chronic RAFTK activation in cardiac remodeling may represent a maladaptive reactive response that can be modulated by paxillin, opening up novel possibilities for inhibition of cardiomyocyte apoptosis and structural degeneration in heart failure.

  8. Farnesoid X Receptor Antagonizes JNK Signaling Pathway in Liver Carcinogenesis by Activating SOD3

    PubMed Central

    Li, Cunbao; Guo, Cong; Li, Yanyan; Qi, Hui; Shen, Hailing; Kong, Jing; Long, Xuecheng; Yuan, Frank; Wang, Xichun

    2015-01-01

    The farnesoid X receptor (FXR) is a key metabolic and homeostatic regulator in the liver. In the present work, we identify a novel role of FXR in antagonizing c-Jun N-terminal kinase (JNK) signaling pathway in liver carcinogenesis by activating superoxide dismutase 3 (SOD3) transcription. Compared with wild-type mouse liver, FXR−/− mouse liver showed elevated JNK phosphorylation. JNK1 deletion suppressed the increase of diethylnitrosamine-induced tumor number in FXR−/− mice. These results suggest that JNK1 plays a key role in chemical-induced liver carcinogenesis in FXR−/− mice. We found that ligand-activated FXR was able to alleviate H2O2 or tetradecanoylphorbol acetate-induced JNK phosphorylation in human hepatoblastoma (HepG2) cells or mouse primary hepatocytes. FXR ligand decreased H2O2-induced reactive oxygen species (ROS) levels in wild-type but not FXR−/− mouse hepatocytes. FXR knockdown abolished the inhibition of 3-[2-[2-chloro-4-[[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl]-Benzoic acid (GW4064) on JNK phosphorylation and ROS production induced by H2O2 in HepG2 cells. The gene expression of SOD3, an antioxidant defense enzyme, was increased by FXR activation in vitro and in vivo. An FXR-responsive element, inverted repeat separated by 1 nucleotide in SOD3 promoter, was identified by a combination of transcriptional reporter assays, EMSAs, and chromatin immunoprecipitation assays, which indicated that SOD3 could be a direct FXR target gene. SOD3 knockdown abolished the inhibition of GW4064 on JNK phosphorylation induced by H2O2 in HepG2 cells. In summary, FXR may regulate SOD3 expression to suppress ROS production, resulting in decreasing JNK activity. These results suggest that FXR, as a novel JNK suppressor, may be an attractive therapeutic target for liver cancer treatment. PMID:25496033

  9. Farnesoid X receptor antagonizes JNK signaling pathway in liver carcinogenesis by activating SOD3.

    PubMed

    Wang, Yan-Dong; Chen, Wei-Dong; Li, Cunbao; Guo, Cong; Li, Yanyan; Qi, Hui; Shen, Hailing; Kong, Jing; Long, Xuecheng; Yuan, Frank; Wang, Xichun; Huang, Wendong

    2015-02-01

    The farnesoid X receptor (FXR) is a key metabolic and homeostatic regulator in the liver. In the present work, we identify a novel role of FXR in antagonizing c-Jun N-terminal kinase (JNK) signaling pathway in liver carcinogenesis by activating superoxide dismutase 3 (SOD3) transcription. Compared with wild-type mouse liver, FXR(-/-) mouse liver showed elevated JNK phosphorylation. JNK1 deletion suppressed the increase of diethylnitrosamine-induced tumor number in FXR(-/-) mice. These results suggest that JNK1 plays a key role in chemical-induced liver carcinogenesis in FXR(-/-) mice. We found that ligand-activated FXR was able to alleviate H₂O₂or tetradecanoylphorbol acetate-induced JNK phosphorylation in human hepatoblastoma (HepG2) cells or mouse primary hepatocytes. FXR ligand decreased H₂O₂-induced reactive oxygen species (ROS) levels in wild-type but not FXR(-/-) mouse hepatocytes. FXR knockdown abolished the inhibition of 3-[2-[2-chloro-4-[[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl]-Benzoic acid (GW4064) on JNK phosphorylation and ROS production induced by H₂O₂in HepG2 cells. The gene expression of SOD3, an antioxidant defense enzyme, was increased by FXR activation in vitro and in vivo. An FXR-responsive element, inverted repeat separated by 1 nucleotide in SOD3 promoter, was identified by a combination of transcriptional reporter assays, EMSAs, and chromatin immunoprecipitation assays, which indicated that SOD3 could be a direct FXR target gene. SOD3 knockdown abolished the inhibition of GW4064 on JNK phosphorylation induced by H₂O₂in HepG2 cells. In summary, FXR may regulate SOD3 expression to suppress ROS production, resulting in decreasing JNK activity. These results suggest that FXR, as a novel JNK suppressor, may be an attractive therapeutic target for liver cancer treatment.

  10. 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

  11. Non-canonical antagonism of PI3K by the kinase Itpkb delays thymocyte β-selection and renders it Notch-dependent

    PubMed Central

    Westernberg, Luise; Conche, Claire; Huang, Yina Hsing; Rigaud, Stephanie; Deng, Yisong; Siegemund, Sabine; Mukherjee, Sayak; Nosaka, Lyn'Al; Das, Jayajit; Sauer, Karsten

    2016-01-01

    β-selection is the most pivotal event determining αβ T cell fate. Here, surface-expression of a pre-T cell receptor (pre-TCR) induces thymocyte metabolic activation, proliferation, survival and differentiation. Besides the pre-TCR, β-selection also requires co-stimulatory signals from Notch receptors - key cell fate determinants in eukaryotes. Here, we show that this Notch-dependence is established through antagonistic signaling by the pre-TCR/Notch effector, phosphoinositide 3-kinase (PI3K), and by inositol-trisphosphate 3-kinase B (Itpkb). Canonically, PI3K is counteracted by the lipid-phosphatases Pten and Inpp5d/SHIP-1. In contrast, Itpkb dampens pre-TCR induced PI3K/Akt signaling by producing IP4, a soluble antagonist of the Akt-activating PI3K-product PIP3. Itpkb-/- thymocytes are pre-TCR hyperresponsive, hyperactivate Akt, downstream mTOR and metabolism, undergo an accelerated β-selection and can develop to CD4+CD8+ cells without Notch. This is reversed by inhibition of Akt, mTOR or glucose metabolism. Thus, non-canonical PI3K-antagonism by Itpkb restricts pre-TCR induced metabolic activation to enforce coincidence-detection of pre-TCR expression and Notch-engagement. DOI: http://dx.doi.org/10.7554/eLife.10786.001 PMID:26880557

  12. Focal adhesion kinase antagonizes doxorubicin cardiotoxicity via p21(Cip1.).

    PubMed

    Cheng, Zhaokang; DiMichele, Laura A; Rojas, Mauricio; Vaziri, Cyrus; Mack, Christopher P; Taylor, Joan M

    2014-02-01

    Clinical application of potent anthracycline anticancer drugs, especially doxorubicin (DOX), is limited by a toxic cardiac side effect that is not fully understood and preventive strategies are yet to be established. Studies in genetically modified mice have demonstrated that focal adhesion kinase (FAK) plays a key role in regulating adaptive responses of the adult myocardium to pathological stimuli through activation of intracellular signaling cascades that facilitate cardiomyocyte growth and survival. The objective of this study was to determine if targeted myocardial FAK activation could protect the heart from DOX-induced de-compensation and to characterize the underlying mechanisms. To this end, mice with myocyte-restricted FAK knock-out (MFKO) or myocyte-specific expression of an active FAK variant (termed SuperFAK) were subjected to DOX treatment. FAK depletion enhanced susceptibility to DOX-induced myocyte apoptosis and cardiac dysfunction, while elevated FAK activity provided remarkable cardioprotection. Our mec6hanistic studies reveal a heretofore unappreciated role for the protective cyclin-dependent kinase inhibitor p21 in the repression of the pro-apoptotic BH3-only protein Bim and the maintenance of mitochondrial integrity and myocyte survival. DOX treatment induced proteasomal degradation of p21, which exacerbated mitochondrial dysfunction and cardiomyocyte apoptosis. FAK was both necessary and sufficient for maintaining p21 levels following DOX treatment and depletion of p21 compromised FAK-dependent protection from DOX. These findings identify p21 as a key determinant of DOX resistance downstream of FAK in cardiomyocytes and indicate that cardiac-restricted enhancement of the FAK/p21 signaling axis might be an effective strategy to preserve myocardial function in patients receiving anthracycline chemotherapy. PMID:24342076

  13. Human Cytomegalovirus pTRS1 and pIRS1 Antagonize Protein Kinase R To Facilitate Virus Replication

    PubMed Central

    Ziehr, Benjamin; Vincent, Heather A.

    2016-01-01

    ABSTRACT Human cytomegalovirus (HCMV) counteracts host defenses that otherwise act to limit viral protein synthesis. One such defense is the antiviral kinase protein kinase R (PKR), which inactivates the eukaryotic initiation factor 2 (eIF2) translation initiation factor upon binding to viral double-stranded RNAs. Previously, the viral TRS1 and IRS1 proteins were found to antagonize the antiviral kinase PKR outside the context of HCMV infection, and the expression of either pTRS1 or pIRS1 was shown to be necessary for HCMV replication. In this study, we found that expression of either pTRS1 or pIRS1 is necessary to prevent PKR activation during HCMV infection and that antagonism of PKR is critical for efficient viral replication. Consistent with a previous study, we observed decreased overall levels of protein synthesis, reduced viral protein expression, and diminished virus replication in the absence of both pTRS1 and pIRS1. In addition, both PKR and eIF2α were phosphorylated during infection when pTRS1 and pIRS1 were absent. We also found that expression of pTRS1 was both necessary and sufficient to prevent stress granule formation in response to eIF2α phosphorylation. Depletion of PKR prevented eIF2α phosphorylation, rescued HCMV replication and protein synthesis, and reversed the accumulation of stress granules in infected cells. Infection with an HCMV mutant lacking the pTRS1 PKR binding domain resulted in PKR activation, suggesting that pTRS1 inhibits PKR through a direct interaction. Together our results show that antagonism of PKR by HCMV pTRS1 and pIRS1 is critical for viral protein expression and efficient HCMV replication. IMPORTANCE To successfully replicate, viruses must counteract host defenses that limit viral protein synthesis. We have identified inhibition of the antiviral kinase PKR by the viral proteins TRS1 and IRS1 and shown that this is a critical step in HCMV replication. Our results suggest that inhibiting pTRS1 and pIRS1 function or

  14. 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

  15. Coop functions as a corepressor of Pangolin and antagonizes Wingless signaling.

    PubMed

    Song, Haiyun; Goetze, Sandra; Bischof, Johannes; Spichiger-Haeusermann, Chloe; Kuster, Marco; Brunner, Erich; Basler, Konrad

    2010-05-01

    Wingless (Wg) signaling regulates expression of its target genes via Pangolin and Armadillo, and their interacting cofactors. In the absence of Wg, Pangolin mediates transcriptional repression. In the presence of Wg, Pangolin, Armadillo, and a cohort of coactivators mediate transcriptional activation. Here we uncover Coop (corepressor of Pan) as a Pangolin-interacting protein. Coop and Pangolin form a complex on DNA containing a Pangolin/TCF-binding motif. Overexpression of Coop specifically represses Wg target genes, while loss of Coop function causes derepression. Finally, we show that Coop antagonizes the binding of Armadillo to Pangolin, providing a mechanism for Coop-mediated repression of Wg target gene transcription. PMID:20439429

  16. Coop functions as a corepressor of Pangolin and antagonizes Wingless signaling.

    PubMed

    Song, Haiyun; Goetze, Sandra; Bischof, Johannes; Spichiger-Haeusermann, Chloe; Kuster, Marco; Brunner, Erich; Basler, Konrad

    2010-05-01

    Wingless (Wg) signaling regulates expression of its target genes via Pangolin and Armadillo, and their interacting cofactors. In the absence of Wg, Pangolin mediates transcriptional repression. In the presence of Wg, Pangolin, Armadillo, and a cohort of coactivators mediate transcriptional activation. Here we uncover Coop (corepressor of Pan) as a Pangolin-interacting protein. Coop and Pangolin form a complex on DNA containing a Pangolin/TCF-binding motif. Overexpression of Coop specifically represses Wg target genes, while loss of Coop function causes derepression. Finally, we show that Coop antagonizes the binding of Armadillo to Pangolin, providing a mechanism for Coop-mediated repression of Wg target gene transcription.

  17. UV-B detected by the UVR8 photoreceptor antagonizes auxin signaling and plant shade avoidance.

    PubMed

    Hayes, Scott; Velanis, Christos N; Jenkins, Gareth I; Franklin, Keara A

    2014-08-12

    Plants detect different facets of their radiation environment via specific photoreceptors to modulate growth and development. UV-B is perceived by the photoreceptor UV RESISTANCE LOCUS 8 (UVR8). The molecular mechanisms linking UVR8 activation to plant growth are not fully understood, however. When grown in close proximity to neighboring vegetation, shade-intolerant plants initiate dramatic stem elongation to overtop competitors. Here we show that UV-B, detected by UVR8, provides an unambiguous sunlight signal that inhibits shade avoidance responses in Arabidopsis thaliana by antagonizing the phytohormones auxin and gibberellin. UV-B triggers degradation of the transcription factors PHYTOCHROME INTERACTING FACTOR 4 and PHYTOCHROME INTERACTING FACTOR 5 and stabilizes growth-repressing DELLA proteins, inhibiting auxin biosynthesis via a dual mechanism. Our findings show that UVR8 signaling is closely integrated with other photoreceptor pathways to regulate auxin signaling and plant growth in sunlight.

  18. PPM1A Regulates Antiviral Signaling by Antagonizing TBK1-Mediated STING Phosphorylation and Aggregation

    PubMed Central

    Li, Zexing; Liu, Ge; Sun, Liwei; Teng, Yan; Guo, Xuejiang; Jia, Jianhang; Sha, Jiahao; Yang, Xiao; Chen, Dahua; Sun, Qinmiao

    2015-01-01

    Stimulator of interferon genes (STING, also known as MITA and ERIS) is critical in protecting the host against DNA pathogen invasion. However, the molecular mechanism underlying the regulation of STING remains unclear. Here, we show that PPM1A negatively regulates antiviral signaling by targeting STING in its phosphatase activity-dependent manner, and in a line with this, PPM1A catalytically dephosphorylates STING and TBK1 in vitro. Importantly, we provide evidence that whereas TBK1 promotes STING aggregation in a phosphorylation-dependent manner, PPM1A antagonizes STING aggregation by dephosphorylating both STING and TBK1, emphasizing that phosphorylation is crucial for the efficient activation of STING. Our findings demonstrate a novel regulatory circuit in which STING and TBK1 reciprocally regulate each other to enable efficient antiviral signaling activation, and PPM1A dephosphorylates STING and TBK1, thereby balancing this antiviral signal transduction. PMID:25815785

  19. 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

  20. Membrane-associated transcription factor peptidase, site-2 protease, antagonizes ABA signaling in Arabidopsis.

    PubMed

    Zhou, Shun-Fan; Sun, Le; Valdés, Ana Elisa; Engström, Peter; Song, Ze-Ting; Lu, Sun-Jie; Liu, Jian-Xiang

    2015-10-01

    Abscisic acid plays important roles in maintaining seed dormancy while gibberellins (GA) and other phytohormones antagonize ABA to promote germination. However, how ABA signaling is desensitized during the transition from dormancy to germination is still poorly understood. We functionally characterized the role of membrane-associated transcription factor peptidase, site-2 protease (S2P), in ABA signaling during seed germination in Arabidopsis. Genetic analysis showed that loss-of-function of S2P conferred high ABA sensitivity during seed germination, and expression of the activated form of membrane-associated transcription factor bZIP17, in which the transmembrane domain and endoplasmic reticulum (ER) lumen-facing C-terminus were deleted, in the S2P mutant rescued its ABA-sensitive phenotype. MYC and green fluorescent protein (GFP)-tagged bZIP17 were processed and translocated from the ER to the nucleus in response to ABA treatment. Furthermore, genes encoding negative regulators of ABA signaling, such as the transcription factor ATHB7 and its target genes HAB1, HAB2, HAI1 and AHG3, were up-regulated in seeds of the wild-type upon ABA treatment; this up-regulation was impaired in seeds of S2P mutants. Our results suggest that S2P desensitizes ABA signaling during seed germination through regulating the activation of the membrane-associated transcription factor bZIP17 and therefore controlling the expression level of genes encoding negative regulators of ABA signaling. PMID:25919792

  1. 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

  2. LIMB DEFECTS INDUCED BY RETINOIC ACID SIGNALING ANTAGONISM AND SYNTHESIS INHIBITION ARE CONSISTENT WITH ETHANOL-INDUCED LIMB DEFECTS

    EPA Science Inventory

    Limb defects induced by retinoic acid signaling antagonism and synthesis inhibition are consistent with ethanol-induced limb defects

    Johnson CS1, Sulik KK1,2, Hunter, ES III3
    1Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, NC....

  3. Wnt signalling antagonizes stress granule assembly through a Dishevelled-dependent mechanism

    PubMed Central

    Sahoo, Pabitra K.; Murawala, Prayag; Sawale, Pravin T.; Sahoo, Manas R.; Tripathi, Mukesh M.; Gaikwad, Swati R.; Seshadri, Vasudevan; Joseph, Jomon

    2012-01-01

    Summary Cells often respond to diverse environmental stresses by inducing stress granules (SGs) as an adaptive mechanism. SGs are generally assembled as a result of aggregation of mRNAs stalled in a translational pre-initiation complex, mediated by a set of RNA-binding proteins such as G3BP and TIA-1. SGs may serve as triage centres for storage, translation re-initiation or degradation of specific mRNAs. However, the mechanism involved in the modulation of their assembly/disassembly is unclear. Here we report that Wnt signalling negatively regulates SG assembly through Dishevelled (Dvl), a cytoplasmic Wnt effector. Overexpression of Dvl2, an isoform of Dvl, leads to impairment of SG assembly through a DEP domain dependent mechanism. Intriguingly, the Dvl2 mutant K446M, which corresponds to an analogous mutation in Drosophila Dishevelled DEP domain (dsh1) that results in defective PCP pathway, fails to antagonize SG assembly. Furthermore, we show that Dvl2 exerts the antagonistic effect on SG assembly through a mechanism involving Rac1-mediated inhibition of RhoA. Dvl2 interacts with G3BP, a downstream component of Ras signalling involved in SG assembly, and functional analysis suggests a model wherein the Dvl-Rac1-RhoA axis regulates G3BP's SG-nucleating activity. Collectively, these results define an antagonistic effect of Wnt signalling on SG assembly, and reveal a novel role for Wnt/Dvl pathway in the modulation of mRNA functions. PMID:23213403

  4. Message in a bottle: lessons learned from antagonism of STING signalling during RNA virus infection

    PubMed Central

    Maringer, Kevin; Fernandez-Sesma, Ana

    2014-01-01

    STING has emerged in recent years as an important signalling adaptor in the activation of type I interferon responses during infection with DNA viruses and bacteria. An increasing body of evidence suggests that STING also modulates responses to RNA viruses, though the mechanisms remain less clear. In this review, we give a brief overview of the ways in which STING facilitates sensing of RNA viruses. These include modulation of RIG-I-dependent responses through STING's interaction with MAVS, and more speculative mechanisms involving the DNA sensor cGAS and sensing of membrane remodelling events. We then provide an in-depth literature review to summarise the known mechanisms by which RNA viruses of the families Flaviviridae and Coronaviridae evade sensing through STING. Our own work has shown that the NS2B/3 protease complex of the flavivirus dengue virus binds and cleaves STING, and that an inability to degrade murine STING may contribute to host restriction in this virus. We contrast this to the mechanism employed by the distantly related hepacivirus hepatitis C virus, in which STING is bound and inactivated by the NS4B protein. Finally, we discuss STING antagonism in the coronaviruses SARS coronavirus and human coronavirus NL63, which disrupt K63-linked polyubiquitination and dimerisation of STING (both of which are required for STING-mediated activation of IRF-3) via their papain-like proteases. We draw parallels with less-well characterised mechanisms of STING antagonism in related viruses, and place our current knowledge in the context of species tropism restrictions that potentially affect the emergence of new human pathogens. PMID:25212897

  5. Agonism, Antagonism, and Inverse Agonism Bias at the Ghrelin Receptor Signaling.

    PubMed

    M'Kadmi, Céline; Leyris, Jean-Philippe; Onfroy, Lauriane; Galés, Céline; Saulière, Aude; Gagne, Didier; Damian, Marjorie; Mary, Sophie; Maingot, Mathieu; Denoyelle, Séverine; Verdié, Pascal; Fehrentz, Jean-Alain; Martinez, Jean; Banères, Jean-Louis; Marie, Jacky

    2015-11-01

    The G protein-coupled receptor GHS-R1a mediates ghrelin-induced growth hormone secretion, food intake, and reward-seeking behaviors. GHS-R1a signals through Gq, Gi/o, G13, and arrestin. Biasing GHS-R1a signaling with specific ligands may lead to the development of more selective drugs to treat obesity or addiction with minimal side effects. To delineate ligand selectivity at GHS-R1a signaling, we analyzed in detail the efficacy of a panel of synthetic ligands activating the different pathways associated with GHS-R1a in HEK293T cells. Besides β-arrestin2 recruitment and ERK1/2 phosphorylation, we monitored activation of a large panel of G protein subtypes using a bioluminescence resonance energy transfer-based assay with G protein-activation biosensors. We first found that unlike full agonists, Gq partial agonists were unable to trigger β-arrestin2 recruitment and ERK1/2 phosphorylation. Using G protein-activation biosensors, we then demonstrated that ghrelin promoted activation of Gq, Gi1, Gi2, Gi3, Goa, Gob, and G13 but not Gs and G12. Besides, we identified some GHS-R1a ligands that preferentially activated Gq and antagonized ghrelin-mediated Gi/Go activation. Finally, we unambiguously demonstrated that in addition to Gq, GHS-R1a also promoted constitutive activation of G13. Importantly, we identified some ligands that were selective inverse agonists toward Gq but not of G13. This demonstrates that bias at GHS-R1a signaling can occur not only with regard to agonism but also to inverse agonism. Our data, combined with other in vivo studies, may facilitate the design of drugs selectively targeting individual signaling pathways to treat only the therapeutically relevant function.

  6. The novel plant homeodomain protein rhinoceros antagonizes Ras signaling in the Drosophila eye.

    PubMed Central

    Voas, Matthew G; Rebay, Ilaria

    2003-01-01

    The sequential specification of cell fates in the Drosophila eye requires repeated activation of the epidermal growth factor receptor (EGFR)/Ras/MAP kinase (MAPK) pathway. Equally important are the multiple layers of inhibitory regulation that prevent excessive or inappropriate signaling. Here we describe the molecular and genetic analysis of a previously uncharacterized gene, rhinoceros (rno), that we propose functions to restrict EGFR signaling in the eye. Loss of rno results in the overproduction of photoreceptors, cone cells, and pigment cells and a corresponding reduction in programmed cell death, all phenotypes characteristic of hyperactivated EGFR signaling. Genetic interactions between rno and multiple EGFR pathway components support this hypothesis. rno encodes a novel but evolutionarily conserved nuclear protein with a PHD zinc-finger domain, a motif commonly found in chromatin-remodeling factors. Future analyses of rno will help to elucidate the regulatory strategies that modulate EGFR signaling in the fly eye. PMID:14704181

  7. Clinical Relevance and Mechanisms of Antagonism Between the BMP and Activin/TGF-β Signaling Pathways.

    PubMed

    Hudnall, Aaron M; Arthur, Jon W; Lowery, Jonathan W

    2016-07-01

    The transforming growth factor β (TGF-β) superfamily is a large group of signaling molecules that participate in embryogenesis, organogenesis, and tissue homeostasis. These molecules are present in all animal genomes. Dysfunction in the regulation or activity of this superfamily's components underlies numerous human diseases and developmental defects. There are 2 distinct arms downstream of the TGF-β superfamily ligands-the bone morphogenetic protein (BMP) and activin/TGF-β signaling pathways-and these 2 responses can oppose one another's effects, most notably in disease states. However, studies have commonly focused on a single arm of the TGF-β superfamily, and the antagonism between these pathways is unknown in most physiologic and pathologic contexts. In this review, the authors summarize the clinically relevant scenarios in which the BMP and activin/TGF-β pathways reportedly oppose one another and identify several molecular mechanisms proposed to mediate this interaction. Particular attention is paid to experimental findings that may be informative to human pathology to highlight potential therapeutic approaches for future investigation. PMID:27367950

  8. Soluble CD109 binds TGF-β and antagonizes TGF-β signalling and responses.

    PubMed

    Li, Carter; Hancock, Mark A; Sehgal, Priyanka; Zhou, Shufeng; Reinhardt, Dieter P; Philip, Anie

    2016-03-01

    Transforming growth factor-β (TGF-β) is a multifunctional cytokine implicated in many diseases, including tissue fibrosis and cancer. TGF-β mediates diverse biological responses by signalling through type I and II TGF-β receptors (TβRI and TβRII). We have previously identified CD109, a glycosylphosphatidylinositol (GPI)-anchored protein, as a novel TGF-β co-receptor that negatively regulates TGF-β signalling and responses and demonstrated that membrane-anchored CD109 promotes TGF-β receptor degradation via a SMAD7/Smurf2-mediated mechanism. To determine whether CD109 released from the cell surface (soluble CD109 or sCD109) also acts as a TGF-β antagonist, we determined the efficacy of recombinant sCD109 to interact with TGF-β and inhibit TGF-β signalling and responses. Our results demonstrate that sCD109 binds TGF-β with high affinity as determined by surface plasmon resonance (SPR) and cell-based radioligand binding and affinity labelling competition assays. SPR detected slow dissociation kinetics between sCD109 and TGF-β at low concentrations, indicating a stable and effective interaction. In addition, sCD109 antagonizes TGF-β-induced Smad2/3 phosphorylation, transcription and cell migration. Together, our results suggest that sCD109 can bind TGF-β, inhibit TGF-β binding to its receptors and decrease TGF-β signalling and TGF-β-induced cellular responses.

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

    PubMed

    Cappell, Steven D; Dohlman, Henrik G

    2011-04-29

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

  10. Brd4 shields chromatin from ATM kinase signaling storms.

    PubMed

    Choi, Serah; Bakkenist, Christopher J

    2013-09-17

    Upon activation, ataxia telangiectasia mutated (ATM) kinase rapidly phosphorylates hundreds of proteins, setting off chaotic signaling storms from areas of damaged chromatin. Recent work by Kaidi and Jackson and Floyd et al. advance our knowledge of the mechanisms that initiate or limit ATM kinase signaling storms at chromatin. PMID:24045152

  11. Brd4 Shields Chromatin from ATM Kinase Signaling Storms

    PubMed Central

    Choi, Serah; Bakkenist, Christopher J.

    2014-01-01

    Upon activation, ataxia telangiectasia mutated (ATM) kinase rapidly phosphorylates hundreds of proteins, setting off chaotic signaling storms from areas of damaged chromatin. Recent work by Kaidi and Jackson and Floyd et al. advance our knowledge of the mechanisms that initiate or limit ATM kinase signaling storms at chromatin. PMID:24045152

  12. A type III effector antagonizes death receptor signalling during bacterial gut infection.

    PubMed

    Pearson, Jaclyn S; Giogha, Cristina; Ong, Sze Ying; Kennedy, Catherine L; Kelly, Michelle; Robinson, Keith S; Lung, Tania Wong Fok; Mansell, Ashley; Riedmaier, Patrice; Oates, Clare V L; Zaid, Ali; Mühlen, Sabrina; Crepin, Valerie F; Marches, Olivier; Ang, Ching-Seng; Williamson, Nicholas A; O'Reilly, Lorraine A; Bankovacki, Aleksandra; Nachbur, Ueli; Infusini, Giuseppe; Webb, Andrew I; Silke, John; Strasser, Andreas; Frankel, Gad; Hartland, Elizabeth L

    2013-09-12

    Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.

  13. SIAH2 antagonizes TYK2-STAT3 signaling in lung carcinoma cells.

    PubMed

    Müller, Sylvia; Chen, Yuan; Ginter, Torsten; Schäfer, Claudia; Buchwald, Marc; Schmitz, Lienhard M; Klitzsch, Jana; Schütz, Alexander; Haitel, Andrea; Schmid, Katharina; Moriggl, Richard; Kenner, Lukas; Friedrich, Karlheinz; Haan, Claude; Petersen, Iver; Heinzel, Thorsten; Krämer, Oliver H

    2014-05-30

    The Janus tyrosine kinases JAK1-3 and tyrosine kinase-2 (TYK2) are frequently hyperactivated in tumors. In lung cancers JAK1 and JAK2 induce oncogenic signaling through STAT3. A putative role of TYK2 in these tumors has not been reported. Here, we show a previously not recognized TYK2-STAT3 signaling node in lung cancer cells. We reveal that the E3 ubiquitin ligase seven-in-absentia-2 (SIAH2) accelerates the proteasomal degradation of TYK2. This mechanism consequently suppresses the activation of STAT3. In agreement with these data the analysis of primary non-small-cell lung cancer (NSCLC) samples from three patient cohorts revealed that compared to lung adenocarcinoma (ADC), lung squamous cell carcinoma (SCC) show significantly higher levels of SIAH2 and reduced STAT3 phosphorylation levels. Thus, SIAH2 is a novel molecular marker for SCC. We further demonstrate that an activation of the oncologically relevant transcription factor p53 in lung cancer cells induces SIAH2, depletes TYK2, and abrogates the tyrosine phosphorylation of STAT1 and STAT3. This mechanism appears to be different from the inhibition of phosphorylated JAKs through the suppressor of cytokine signaling (SOCS) proteins. Our study may help to identify molecular mechanisms affecting lung carcinogenesis and potential therapeutic targets. PMID:24833526

  14. Exosome release of β-catenin: a novel mechanism that antagonizes Wnt signaling

    PubMed Central

    Chairoungdua, Arthit; Smith, Danielle L.; Pochard, Pierre; Hull, Michael

    2010-01-01

    CD82 and CD9 are tetraspanin membrane proteins that can function as suppressors of tumor metastasis. Expression of CD9 and CD82 in transfected cells strongly suppresses β-catenin–mediated Wnt signaling activity and induces a significant decrease in β-catenin protein levels. Inhibition of Wnt/β-catenin signaling is independent of glycogen synthase kinase-3β and of the proteasome- and lysosome-mediated protein degradation pathways. CD82 and CD9 expression induces β-catenin export via exosomes, which is blocked by a sphingomyelinase inhibitor, GW4869. CD82 fails to induce exosome release of β-catenin in cells that express low levels of E-cadherin. Exosome release from dendritic cells generated from CD9 knockout mice is reduced compared with that from wild-type dendritic cells. These results suggest that CD82 and CD9 down-regulate the Wnt signaling pathway through the exosomal discharge of β-catenin. Thus, exosomal packaging and release of cytosolic proteins can modulate the activity of cellular signaling pathways. PMID:20837771

  15. 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

  16. 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

  17. Regulation of Wnt/β-Catenin Signaling by Protein Kinases

    PubMed Central

    Verheyen, Esther M.; Gottardi, Cara J.

    2011-01-01

    The Wnt/β-catenin signaling pathway plays essential roles during development and adult tissue homeostasis. Inappropriate activation of the pathway can result in a variety of malignancies. Protein kinases have emerged as key regulators at multiple steps of the Wnt pathway. In this review, we present a synthesis covering the latest information on how Wnt signaling is regulated by diverse protein kinases. PMID:19623618

  18. DAF-18/PTEN locally antagonizes insulin signalling to couple germline stem cell proliferation to oocyte needs in C. elegans.

    PubMed

    Narbonne, Patrick; Maddox, Paul S; Labbé, Jean-Claude

    2015-12-15

    During development, stem cell populations rapidly proliferate to populate the expanding tissues and organs. During this phase, nutrient status, by systemically affecting insulin/IGF-1 signalling, largely dictates stem cell proliferation rates. In adults, however, differentiated stem cell progeny requirements are generally reduced and vary according to the spatiotemporal needs of each tissue. We demonstrate here that differential regulation of germline stem cell proliferation rates in Caenorhabditis elegans adults is accomplished through localized neutralization of insulin/IGF-1 signalling, requiring DAF-18/PTEN, but not DAF-16/FOXO. Indeed, the specific accumulation of oocytes, the terminally differentiated stem cell progeny, triggers a feedback signal that locally antagonizes insulin/IGF-1 signalling outputs in the germ line, regardless of their systemic levels, to block germline stem cell proliferation. Thus, during adulthood, stem cells can differentially respond within tissues to otherwise equal insulin/IGF-1 signalling inputs, according to the needs for production of their immediate terminally differentiated progeny.

  19. 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

  20. 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.

  1. 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

  2. Perspective: Dynamics of receptor tyrosine kinase signaling complexes.

    PubMed

    Mayer, Bruce J

    2012-08-14

    Textbook descriptions of signal transduction complexes provide a static snapshot view of highly dynamic events. Despite enormous strides in identifying the key components of signaling complexes and the underlying mechanisms of signal transduction, our understanding of the dynamic behavior of these complexes has lagged behind. Using the example of receptor tyrosine kinases, this perspective takes a fresh look at the dynamics of the system and their potential impact on signal processing. PMID:22584051

  3. PIM kinase (and Akt) biology and signaling in tumors

    PubMed Central

    Warfel, Noel A.; Kraft, Andrew S.

    2016-01-01

    The initiation and progression of human cancer is frequently linked to the uncontrolled activation of survival kinases. Two such pro-survival kinases that are commonly amplified in cancer are PIM and Akt. These oncogenic proteins are serine/threonine kinases that regulate tumorigenesis by phosphorylating substrates that control the cell cycle, cellular metabolism, proliferation, and survival. Growing evidence suggests that cross-talk exists between the PIM and Akt kinases, indicating that they control partially overlapping survival signaling pathways that are critical to the initiation, progression, and metastatic spread of many types of cancer. The PI3K/Akt signaling pathway is activated in many human tumors, and it is well established as a promising anticancer target. Likewise, based on the role of PIM kinases in normal and tumor tissues, it is clear that this family of kinases represents an interesting target for anticancer therapy. Pharmacological inhibition of PIM has the potential to significantly influence the efficacy of standard and targeted therapies. This review focuses on the regulation of PIM kinases, their role in tumorigenesis, and the biological impact of their interaction with the Akt signaling pathway on the efficacy of cancer therapy. PMID:25749412

  4. Mitogen-activated protein kinase and abscisic acid signal transduction.

    PubMed

    Heimovaara-Dijkstra, S; Testerink, C; Wang, M

    2000-01-01

    The phytohormone abscisic acid (ABA) is a classical plant hormone, responsible for regulation of abscission, diverse aspects of plant and seed development, stress responses and germination. It was found that ABA signal transduction in plants can involve the activity of type 2C-phosphatases (PP2C), calcium, potassium, pH and a transient activation of MAP kinase. The ABA signal transduction cascades have been shown to be tissue-specific, the transient activation of MAP kinase has until now only been found in barley aleurone cells. However, type 2C phosphatases are involved in the induction of most ABA responses, as shown by the PP2C-deficient abi-mutants. These phosphatases show high homology with phosphatases that regulate MAP kinase activity in yeast. In addition, the role of farnesyl transferase as a negative regulator of ABA responses also indicates towards involvement of MAP kinase in ABA signal transduction. Farnesyl transferase is known to regulate Ras proteins, Ras proteins in turn are known to regulate MAP kinase activation. Interestingly, Ras-like proteins were detected in barley aleurone cells. Further establishment of the involvement of MAP kinase in ABA signal transduction and its role therein, still awaits more study.

  5. 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...

  6. Tyrosine kinase signaling and the emergence of multicellularity.

    PubMed

    Miller, W Todd

    2012-06-01

    Tyrosine phosphorylation is an essential element of signal transduction in multicellular animals. Although tyrosine kinases were originally regarded as specific to the metazoan lineage, it is now clear that they evolved prior to the split between unicellular and multicellular eukaryotes (≈600million years ago). Genome analyses of choanoflagellates and other protists show an abundance of tyrosine kinases that rivals the most complex animals. Some of these kinases are orthologs of metazoan enzymes (e.g., Src), but others display unique domain compositions not seen in any metazoan. Biochemical experiments have highlighted similarities and differences between the unicellular and multicellular tyrosine kinases. In particular, it appears that the complex systems of kinase autoregulation may have evolved later in the metazoan lineage. PMID:22480439

  7. Tyrosine kinase signaling and the emergence of multicellularity.

    PubMed

    Miller, W Todd

    2012-06-01

    Tyrosine phosphorylation is an essential element of signal transduction in multicellular animals. Although tyrosine kinases were originally regarded as specific to the metazoan lineage, it is now clear that they evolved prior to the split between unicellular and multicellular eukaryotes (≈600million years ago). Genome analyses of choanoflagellates and other protists show an abundance of tyrosine kinases that rivals the most complex animals. Some of these kinases are orthologs of metazoan enzymes (e.g., Src), but others display unique domain compositions not seen in any metazoan. Biochemical experiments have highlighted similarities and differences between the unicellular and multicellular tyrosine kinases. In particular, it appears that the complex systems of kinase autoregulation may have evolved later in the metazoan lineage.

  8. Calcium-Dependent Signaling and Kinases in Apicomplexan Parasites

    PubMed Central

    Billker, Oliver; Lourido, Sebastian; Sibley, L. David

    2009-01-01

    Summary Calcium controls many critical events in the complex life cycles of apicomplexan parasites including protein secretion, motility, and development. Calcium levels are normally tightly regulated and rapid release of calcium into the cytosol activates a family of calcium-dependent protein kinases (CDPKs), which are normally characteristic of plants. CDPKs present in apicomplexans have acquired a number of unique domain structures likely reflecting their diverse functions. Calcium regulation in parasites is closely linked to signaling by cyclic nucleotides and their associated kinases. This review summarizes the pivotal roles that calcium-and cyclic nucleotide-dependent kinases play in unique aspects of parasite biology. PMID:19527888

  9. 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

  10. The Role of Phosphoinositide 3-Kinase Signaling in Intestinal Inflammation

    PubMed Central

    Cahill, Catherine M.; Rogers, Jack T.; Walker, W. Allan

    2012-01-01

    The phosphatidylinositol 3-kinase signaling pathway plays a central role in regulating the host inflammatory response. The net effect can either be pro- or anti-inflammatory depending on the system and cellular context studied. This paper focuses on phosphatidylinositol 3-kinase signaling in innate and adaptive immune cells of the intestinal mucosa. The role of phosphatidylinositol 3-kinase signaling in mouse models of inflammatory bowel disease is also discussed. With the development of new isoform specific inhibitors, we are beginning to understand the specific role of this complex pathway, in particular the role of the γ isoform in intestinal inflammation. Continued research on this complex pathway will enhance our understanding of its role and provide rationale for the design of new approaches to intervention in chronic inflammatory conditions such as inflammatory bowel disease. PMID:22570785

  11. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling.

    PubMed

    Abedini, Atefeh; Zamberlam, Gustavo; Lapointe, Evelyne; Tourigny, Catherine; Boyer, Alexandre; Paquet, Marilène; Hayashi, Kanako; Honda, Hiroaki; Kikuchi, Akira; Price, Christopher; Boerboom, Derek

    2016-04-01

    Whereas the roles of the canonical wingless-type MMTV (mouse mammary tumor virus) integration site family (WNT) signaling pathway in the regulation of ovarian follicle growth and steroidogenesis are now established, noncanonical WNT signaling in the ovary has been largely overlooked. Noncanonical WNTs, including WNT5a and WNT11, are expressed in granulosa cells (GCs) and are differentially regulated throughout follicle development, but their physiologic roles remain unknown. Using conditional gene targeting, we found that GC-specific inactivation ofWnt5a(but notWnt11) results in the female subfertility associated with increased follicular atresia and decreased rates of ovulation. Microarray analyses have revealed that WNT5a acts to down-regulate the expression of FSH-responsive genesin vitro, and corresponding increases in the expression of these genes have been found in the GCs of conditional knockout mice. Unexpectedly, we found that WNT5a regulates its target genes not by signalingviathe WNT/Ca(2+)or planar cell polarity pathways, but rather by inhibiting the canonical pathway, causing both β-catenin (CTNNB1) and cAMP responsive element binding (CREB) protein levels to decreaseviaa glycogen synthase kinase-3β-dependent mechanism. We further found that WNT5a prevents follicle-stimulating hormone and luteinizing protein from up-regulating the CTNNB1 and CREB proteins and their target genes, indicating that WNT5a functions as a physiologic inhibitor of gonadotropin signaling. Together, these findings identify WNT5a as a key regulator of follicle development and gonadotropin responsiveness.-Abedini, A., Zamberlam, G., Lapointe, E., Tourigny, C., Boyer, A., Paquet, M., Hayashi, K., Honda, H., Kikuchi, A., Price, C., Boerboom, D. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling. PMID:26667040

  12. NAF-1 antagonizes starvation-induced autophagy through AMPK signaling pathway in cardiomyocytes.

    PubMed

    Du, Xiaohong; Xiao, Renjie; Xiao, Fan; Chen, Yong; Hua, Fuzhou; Yu, Shuchun; Xu, Guohai

    2015-07-01

    NAF-1 (nutrient-deprivation autophagy factor-1), an autophagy-related gene-related (ATG) protein, has been implicated in the autophagic pro-survival response. However, its role in autophagy has not been examined in the cardiomyocytes. In this study, we found that nutritional stress (NS) induced by glucose deprivation strongly induced autophagy in cultured neonatal rat cardiomyocytes, which was associated with NAF-1 down-regulation in cardiomyocytes under NS conditions. Furthermore, we demonstrate that ectopic expression of NAF-1 was sufficient to inhibit autophagy in cardiomyocytes under glucose deprivation conditions. Moreover, results of the co-immunoprecipitation assay indicate that NAF-1 antagonized autophagy by promoting the interaction between Beclin1 and Bcl-2 in NS-induced cardiomyocytes. Importantly, our results indicate that overexpression of NAF-1 significantly inhibited AMPK activity and protected cardiomyocytes from NS-induced cell death. Taken together, these data show that ectopic expression of NAF-1 antagonizes the degree of autophagy in cardiomyocytes and enhances cell survival during starvation conditions.

  13. Resveratrol inhibits epithelial-mesenchymal transition and renal fibrosis by antagonizing the hedgehog signaling pathway.

    PubMed

    Bai, Yongheng; Lu, Hong; Wu, Cunzao; Liang, Yong; Wang, Silu; Lin, Chengcheng; Chen, Bicheng; Xia, Peng

    2014-12-01

    Epithelial-to-mesenchymal transition (EMT), a biologic process in which tubular cells lose their epithelial phenotypes and acquire new characteristic features of mesenchymal properties, is increasingly recognized as an integral part of renal tissue fibrogenesis. Recent studies indicate that resveratrol, a botanical compound derived mainly from the skins of red grapes, may have anti-fibrotic effects in many tissues, but the potential molecular mechanism remains unknown. In the present study, we identified that resveratrol inhibits the induction of EMT and deposition of extracellular matrix (ECM) through antagonizing the hedgehog pathway in vitro and in vivo. In rats with unilateral ureteral obstruction (UUO), administration of resveratrol (20mg/kg/day) significantly reduced serum creatinine. Resveratrol also decreased expression of TGF-β1, and inhibited the phenotypic transition from epithelial cells to mesenchymal cells, and the deposition of ECM in UUO rats. In cultured renal tubular epithelial cells (NRK-52E), TGF-β1-induced EMT and ECM synthesis was abolished with the treatment of resveratrol. The induction of EMT was associated with the activation of the hedgehog pathway. Resveratrol treatment markedly inhibited the over-activity of the hedgehog pathway in the obstructed kidney and in TGF-β1-treated NRK-52E cells, resulted in reduction of cellular proliferation, EMT and ECM accumulation. Thus, these results suggest that resveratrol is able to inhibit EMT and fibrosis in vivo and in vitro through antagonizing the hedgehog pathway, and resveratrol may have therapeutic potential for patients with fibrotic kidney diseases.

  14. New functional activity of aripiprazole revealed: Robust antagonism of D2 dopamine receptor-stimulated Gβγ signaling.

    PubMed

    Brust, Tarsis F; Hayes, Michael P; Roman, David L; Watts, Val J

    2015-01-01

    The dopamine D2 receptor (DRD2) is a G protein-coupled receptor (GPCR) that is generally considered to be a primary target in the treatment of schizophrenia. First generation antipsychotic drugs (e.g. haloperidol) are antagonists of the DRD2, while second generation antipsychotic drugs (e.g. olanzapine) antagonize DRD2 and 5HT2A receptors. Notably, both these classes of drugs may cause side effects associated with D2 receptor antagonism (e.g. hyperprolactemia and extrapyramidal symptoms). The novel, "third generation" antipsychotic drug, aripiprazole is also used to treat schizophrenia, with the remarkable advantage that its tendency to cause extrapyramidal symptoms is minimal. Aripiprazole is considered a partial agonist of the DRD2, but it also has partial agonist/antagonist activity for other GPCRs. Further, aripiprazole has been reported to have a unique activity profile in functional assays with the DRD2. In the present study the molecular pharmacology of aripiprazole was further examined in HEK cell models stably expressing the DRD2 and specific isoforms of adenylyl cyclase to assess functional responses of Gα and Gβγ subunits. Additional studies examined the activity of aripiprazole in DRD2-mediated heterologous sensitization of adenylyl cyclase and cell-based dynamic mass redistribution (DMR). Aripiprazole displayed a unique functional profile for modulation of G proteins, being a partial agonist for Gαi/o and a robust antagonist for Gβγ signaling. Additionally, aripiprazole was a weak partial agonist for both heterologous sensitization and dynamic mass redistribution.

  15. 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

  16. 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

  17. Glutamate signalling via a MEKK1 kinase-dependent pathway induces changes in Arabidopsis root architecture.

    PubMed

    Forde, Brian G; Cutler, Sean R; Zaman, Najia; Krysan, Patrick J

    2013-07-01

    A chemical genetic approach has been used to investigate the mechanism by which external glutamate (l-Glu) is able to trigger major changes in root architecture in Arabidopsis thaliana L. An initial screen of 80 agonists and antagonists of mammalian glutamate and GABA receptors, using a specially developed 96-well microphenotyping system, found none that replicated the response of the root to l-Glu or antagonized it. However, a larger screen using >1500 molecules bioactive in Saccharomyces cerevisiae (yeast) identified two groups that interfered with the l-Glu response. One of the antagonists, 2-(4-chloro-3-methylphenyl)-2-oxoethyl thiocyanate (CMOT), has been reported to target Ste11, an evolutionarily conserved MAP kinase kinase kinase (MAP3K) in yeast. This led to the discovery that root growth in a triple mekk1 mekk2 mekk3 mutant (mekk1/2/3), defective in a set of three tandemly arranged MAP3Ks, was almost insensitive to l-Glu. However, the sensitivity of mekk1/2/3 roots to inhibition by other amino acids reported to act as agonists of glutamate receptor-like (GLR) channels in Arabidopsis roots (Asn, Cys, Gly and Ser) was unaffected. The l-Glu sensitivity of the mekk1/2/3 mutant was restored by transformation with a construct carrying the intact MEKK1 gene. These results demonstrate that MEKK1 plays a key role in transducing the l-Glu signal that elicits large-scale changes in root architecture, and provide genetic evidence for the existence in plants of an l-Glu signalling pathway analogous to that found in animals.

  18. 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.

  19. 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

  20. An interdigit signalling centre instructs coordinate phalanx-joint formation governed by 5′Hoxd–Gli3 antagonism

    PubMed Central

    Huang, Bau-Lin; Trofka, Anna; Furusawa, Aki; Norrie, Jacqueline L.; Rabinowitz, Adam H.; Vokes, Steven A.; Mark Taketo, M.; Zakany, Jozsef; Mackem, Susan

    2016-01-01

    The number of phalanges and joints are key features of digit ‘identity' and are central to limb functionality and evolutionary adaptation. Prior chick work indicated that digit phalanges and their associated joints arise in a different manner than the more sparsely jointed long bones, and their identity is regulated by differential signalling from adjacent interdigits. Currently, there is no genetic evidence for this model, and the molecular mechanisms governing digit joint specification remain poorly understood. Using genetic approaches in mouse, here we show that functional 5′Hoxd–Gli3 antagonism acts indirectly, through Bmp signalling from the interdigital mesenchyme, to regulate specification of joint progenitors, which arise in conjunction with phalangeal precursors at the digit tip. Phalanx number, although co-regulated, can be uncoupled from joint specification. We propose that 5′Hoxd genes and Gli3 are part of an interdigital signalling centre that sets net Bmp signalling levels from different interdigits to coordinately regulate phalanx and joint formation. PMID:27713395

  1. Placental-Specific sFLT-1 e15a Protein Is Increased in Preeclampsia, Antagonizes Vascular Endothelial Growth Factor Signaling, and Has Antiangiogenic Activity.

    PubMed

    Palmer, Kirsten R; Kaitu'u-Lino, Tu'uhevaha J; Hastie, Roxanne; Hannan, Natalie J; Ye, Louie; Binder, Natalie; Cannon, Ping; Tuohey, Laura; Johns, Terrance G; Shub, Alexis; Tong, Stephen

    2015-12-01

    In preeclampsia, the antiangiogenic factor soluble fms-like tyrosine kinase-1 (sFLT-1) is released from placenta into the maternal circulation, causing endothelial dysfunction and organ injury. A recently described splice variant, sFLT-1 e15a, is primate specific and the most abundant placentally derived sFLT-1. Therefore, it may be the major sFLT-1 isoform contributing to the pathophysiology of preeclampsia. sFLT-1 e15a protein remains poorly characterized: its bioactivity has not been comprehensively examined, and serum levels in normal and preeclamptic pregnancy have not been reported. We generated and validated an sFLT-1 e15a-specific ELISA to further characterize serum levels during pregnancy, and in the presence of preeclampsia. Furthermore, we performed assays to examine the bioactivity and antiangiogenic properties of sFLT-1 e15a protein. sFLT-1 e15a was expressed in the syncytiotrophoblast, and serum levels rose across pregnancy. Strikingly, serum levels were increased 10-fold in preterm preeclampsia compared with normotensive controls. We confirmed sFLT-1 e15a is bioactive and is able to inhibit vascular endothelial growth factor signaling of vascular endothelial growth factor receptor 2 and block downstream Akt phosphorylation. Furthermore, sFLT-1 e15a has antiangiogenic properties. sFLT-1 e15a decreased endothelial cell migration, invasion, and inhibited endothelial cell tube formation. Administering sFLT-1 e15a blocked vascular endothelial growth factor induced sprouts from mouse aortic rings ex vivo. We have demonstrated that sFLT-1 e15a is increased in preeclampsia, antagonizes vascular endothelial growth factor signaling, and has antiangiogenic activity. Future development of diagnostics and therapeutics for preeclampsia should consider targeting placentally derived sFLT-1 e15a.

  2. 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

  3. Role of protein kinase D signaling in pancreatic cancer.

    PubMed

    Guha, Sushovan; Tanasanvimon, Suebpong; Sinnett-Smith, James; Rozengurt, Enrique

    2010-12-15

    Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with dismal survival rates. Its intransigence to conventional therapy renders PDAC an aggressive disease with early metastatic potential. Thus, novel targets for PDAC therapy are urgently needed. Multiple signal transduction pathways are implicated in progression of PDAC. These pathways stimulate production of intracellular messengers in their target cells to modify their behavior, including the lipid-derived diacylglycerol (DAG). One of the prominent intracellular targets of DAG is the protein kinase C (PKC) family. However, the mechanisms by which PKC-mediated signals are decoded by the cell remain incompletely understood. Protein kinase D1 (PKD or PKD1, initially called atypical PKCμ), is the founding member of a novel protein kinase family that includes two additional protein kinases that share extensive overall homology with PKD, termed PKD2, and PKD3. The PKD family occupies a unique position in the signal transduction pathways initiated by DAG and PKC. PKD lies downstream of PKCs in a novel signal transduction pathway implicated in the regulation of multiple fundamental biological processes. We and others have shown that PKD-mediated signaling pathways promote mitogenesis and angiogenesis in PDAC. Our recent observations demonstrate that PKD also potentiates chemoresistance and invasive potential of PDAC cells. This review will briefly highlight diverse biological roles of PKD family in multiple neoplasias including PDAC. Further, this review will underscore our latest advancement with the development of a potent PKD family inhibitor and its effect both in vitro and in vivo in PDAC. PMID:20621068

  4. Type 2 Interleukin-4 Receptor Signaling in Neutrophils Antagonizes Their Expansion and Migration during Infection and Inflammation.

    PubMed

    Woytschak, Janine; Keller, Nadia; Krieg, Carsten; Impellizzieri, Daniela; Thompson, Robert W; Wynn, Thomas A; Zinkernagel, Annelies S; Boyman, Onur

    2016-07-19

    Neutrophils are the first immune cells recruited to sites of inflammation and infection. However, patients with allergic disorders such as atopic dermatitis show a paucity of skin neutrophils and are prone to bacterial skin infections, suggesting that allergic inflammation curtails neutrophil responses. Here we have shown that the type 2 cell signature cytokine interleukin-4 (IL-4) hampers neutrophil expansion and migration by antagonizing granulocyte colony-stimulating factor (G-CSF) and chemokine receptor-mediated signals. Cutaneous bacterial infection in mice was exacerbated by IL-4 signaling and improved with IL-4 inhibition, each outcome inversely correlating with neutrophil migration to skin. Likewise, systemic bacterial infection was worsened by heightened IL-4 activity, with IL-4 restricting G-CSF-induced neutrophil expansion and migration to tissues by affecting CXCR2-CXCR4 chemokine signaling in neutrophils. These effects were dependent on IL-4 acting through type 2 IL-4 receptors on neutrophils. Thus, targeting IL-4 might be beneficial in neutropenic conditions with increased susceptibility to bacterial infections. PMID:27438770

  5. FES/FER kinase signaling in hematopoietic cells and leukemias.

    PubMed

    Craig, Andrew W B

    2012-01-01

    FES and FES-related (FER) comprise a unique subfamily of protein-tyrosine kinases (PTKs) that signal downstream of several classes of receptors involved in regulating hematopoietic cell development, survival, migration, and inflammatory mediator release. Activated alleles of FES are potent inducers of myeloid differentiation, however FES-deficient mice have only subtle differences in hematopoiesis. This may reflect overlapping function of other kinases such as FER. Studies of FES- and FER-deficient mice have revealed more prominent roles in regulating the activation of mature innate immune cells, including macrophages and mast cells. Recently, new insights into regulation of FES/FER kinases has emerged with the characterization of their N-terminal phospholipid-binding and membrane targeting FER/CIP4 homology-Bin/Amphyphysin/Rvs (F-BAR) and F-BAR extension (FX) domains. The F-BAR/FX domains regulate subcellular localization and FES/FER kinase activation. FES kinase activity is also enhanced upon ligand binding to its SH2 domain, which may lead to further phosphorylation of the same ligand, or other ligand-associated proteins. In mast cells, SH2 ligands of FES/FER include KIT receptor PTK, and the high affinity IgE receptor (FceRI) that trigger rapid activation of FES/FER and signaling to regulators of the actin cytoskeleton and membrane trafficking. Recently, FES/FER have also been implicated in growth and survival signaling in leukemias driven by oncogenic KIT and FLT3 receptors. With further definition of their roles in immune cells and their progenitors, FES/FER may emerge as relevant therapeutic targets in inflammatory diseases and leukemias.

  6. Signaling Network Map of Endothelial TEK Tyrosine Kinase.

    PubMed

    Khan, Aafaque Ahmad; Sandhya, Varot K; Singh, Priyata; Parthasarathy, Deepak; Kumar, Awinav; Advani, Jayshree; Gattu, Rudrappa; Ranjit, Dhanya V; Vaidyanathan, Rama; Mathur, Premendu Prakash; Prasad, T S Keshava; Mac Gabhann, F; Pandey, Akhilesh; Raju, Rajesh; Gowda, Harsha

    2014-01-01

    TEK tyrosine kinase is primarily expressed on endothelial cells and is most commonly referred to as TIE2. TIE2 is a receptor tyrosine kinase modulated by its ligands, angiopoietins, to regulate the development and remodeling of vascular system. It is also one of the critical pathways associated with tumor angiogenesis and familial venous malformations. Apart from the vascular system, TIE2 signaling is also associated with postnatal hematopoiesis. Despite the involvement of TIE2-angiopoietin system in several diseases, the downstream molecular events of TIE2-angiopoietin signaling are not reported in any pathway repository. Therefore, carrying out a detailed review of published literature, we have documented molecular signaling events mediated by TIE2 in response to angiopoietins and developed a network map of TIE2 signaling. The pathway information is freely available to the scientific community through NetPath, a manually curated resource of signaling pathways. We hope that this pathway resource will provide an in-depth view of TIE2-angiopoietin signaling and will lead to identification of potential therapeutic targets for TIE2-angiopoietin associated disorders.

  7. 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 6-12 h 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 33 h 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

  8. Antagonism or Synergism: Role of Tyrosine Phosphatases SHP-1 and SHP-2 in Growth Factor Signaling

    PubMed Central

    Wang, Ning; Li, Zhe; Ding, Ronghua; Frank, Gerald D.; Senbonmatsu, Takaaki; Landon, Erwin J.; Inagami, Tadashi; Zhao, Zhizhuang Joe

    2008-01-01

    SHP-1 and SHP-2 are two SH2 domain-containing tyrosine phosphatases with major pathological implications in cell growth regulating signaling. They share significant overall sequence identity, but their biological functions are often opposite. SHP-1 is generally considered as a negative signal transducer while SHP-2 as a positive one. However, the precise role of each enzyme in shared signaling pathways is not well defined. In this study, we investigated the interaction of these two enzymes in a single cell system by knocking down their expressions with siRNAs and analyzing the effects on epidermal growth factor signaling. Interestingly, knockdown of either SHP-1 or SHP-2 caused significant reduction in the activation of ERK1/2 but not Akt. Furthermore, SHP-1, SHP-2, and Gab1 formed a signaling complex, and SHP-1 and SHP-2 interact with each other. The interaction of SHP-1 with Gab1 is mediated by SHP-2 since it was abrogated by knockdown of SHP-2 and SHP-2, but not SHP-1, binds directly to tyrosine phosphorylated Gab1. Together, the data revealed that both SHP-1 and SHP-2 have a positive role in epidermal growth factor-induced ERK1/2 activation and that they act cooperatively rather than antagonistically. The interaction of SHP-1 and SHP-2 may be responsible for hitherto unexpected novel regulatory mechanism of cell signaling by tyrosine phosphatases. PMID:16762922

  9. 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

  10. 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

  11. δ-Opioid receptors stimulate the metabolic sensor AMP-activated protein kinase through coincident signaling with G(q/11)-coupled receptors.

    PubMed

    Olianas, Maria C; Dedoni, Simona; Olianas, Alessandra; Onali, Pierluigi

    2012-02-01

    AMP-activated protein kinase (AMPK) and δ-opioid receptors (DORs) are both involved in controlling cell survival, energy metabolism, and food intake, but little is known on the interaction between these two signaling molecules. Here we show that activation of human DORs stably expressed in Chinese hamster ovary (CHO) cells increased AMPK activity and AMPK phosphorylation on Thr172. DOR-induced AMPK phosphorylation was prevented by pertussis toxin, reduced by protein kinase A (PKA) activators, and unaffected by PKA, transforming growth factor-β-activated kinase 1, mitogen-activated protein kinase, and protein kinase C inhibitors. Conversely, the DOR effect was reduced by Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) inhibition, apyrase treatment, G(q/11) antagonism, and blockade of P2 purinergic receptors. Apyrase treatment also depressed DOR stimulation of intracellular Ca(2+) concentration, whereas P2 receptor antagonism blocked DOR stimulation of inositol phosphate accumulation. In SH-SY5Y neuroblastoma cells and primary olfactory bulb neurons, DOR activation failed to affect AMPK phosphorylation per se but potentiated the stimulation by either muscarinic agonists or 2-methyl-thio-ADP. Sequestration of G protein βγ subunits (Gβγ) blocked the DOR potentiation of AMPK phosphorylation induced by oxotremorine-M. In CHO cells, the AMPK activator 5-aminoimidazole-4-carboxamide1-β-D-ribonucleoside stimulated AMPK phosphorylation and glucose uptake, whereas pharmacological inhibition of AMPK, expression of a dominant-negative mutant of AMPKα1, and P2Y receptor blockade reduced DOR-stimulated glucose uptake. The data indicate that in different cell systems, DOR activation up-regulates AMPK through a Gβγ-dependent synergistic interaction with G(q/11)-coupled receptors, potentiating Ca(2+) release and CaMKKβ-dependent AMPK phosphorylation. In CHO cells, this coincident signaling mechanism is involved in DOR-induced glucose uptake. PMID:22031472

  12. Effects of Membrane Trafficking on Signaling by Receptor Tyrosine Kinases

    PubMed Central

    Miaczynska, Marta

    2013-01-01

    The intracellular trafficking machinery contributes to the spatial and temporal control of signaling by receptor tyrosine kinases (RTKs). The primary role in this process is played by endocytic trafficking, which regulates the localization of RTKs and their downstream effectors, as well as the duration and the extent of their activity. The key regulatory points along the endocytic pathway are internalization of RTKs from the plasma membrane, their sorting to degradation or recycling, and their residence in various endosomal compartments. Here I will review factors and mechanisms that modulate RTK signaling by (1) affecting receptor internalization, (2) regulating the balance between degradation and recycling of RTK, and (3) compartmentalization of signals in endosomes and other organelles. Cumulatively, these mechanisms illustrate a multilayered control of RTK signaling exerted by the trafficking machinery. PMID:24186066

  13. FES kinases are required for oncogenic FLT3 signaling.

    PubMed

    Voisset, E; Lopez, S; Chaix, A; Georges, C; Hanssens, K; Prébet, T; Dubreuil, P; De Sepulveda, P

    2010-04-01

    The closely related non-receptor tyrosine kinases FEline Sarcoma (FES) and FEs Related (FER) are activated by cell surface receptors in hematopoietic cells. Despite the early description of oncogenic viral forms of fes, v-fes, and v-fps, the implication of FES and FER in human pathology is not known. We have recently shown that FES but not FER is necessary for oncogenic KIT receptor signaling. Here, we report that both FES and FER kinases are activated in primary acute myeloid leukemia (AML) blasts and in AML cell lines. FES and FER activation is dependent on FLT3 in cell lines harboring constitutively active FLT3 mutants. Moreover, both FES and FER proteins are critical for FLT3-internal tandem duplication (ITD) signaling and for cell proliferation in relevant AML cell lines. FER is required for cell cycle transitions, whereas FES seems necessary for cell survival. We concluded that FES and FER kinases mediate essential non-redundant functions downstream of FLT3-ITD.

  14. ZnRF3 Induces Apoptosis of Gastric Cancer Cells by Antagonizing Wnt and Hedgehog Signaling.

    PubMed

    Qin, Hongzhen; Cai, Aizhen; Xi, Hongqing; Yuan, Jing; Chen, Lin

    2015-11-01

    A large proportion of malignant cancers of the stomach are gastric adenocarcinoma type. In spite of many studies, the molecular basis for this cancer is still unclear. Deregulated cell proliferative signaling via Wnt/β-catenin and Hedgehog pathways is considered important in the pathogenesis of many cancers including the gastric cancer. Recent studies identified ZnRF3 protein, which is a E3-ubiquitin ligase and which is either deleted or mutated in cancers, to inhibit Wnt signaling. However, the significance of ZnRF3 in the control of gastric cancer and whether it also regulates Hedgehog signaling pathway, is not known. In the present study, we assessed the expression of ZnRF3 in gastric tumors and paracancerous tissues from 58 patients (44 male and 14 female) of different ages and related this to patient survival. We observed a clear relationship between ZnRF3 expression in paracancerous tissue and tumor size. Also, ZnRF3 expression was much higher in tumors from aged patients. Male patients showed higher mortality than the females. Mechanistic studies using normal gastric cells (GES1) and gastric cancer cells (MGC-803) infected with either AdZnRF3 or AdGFP viral vectors, revealed that ZnRF3 overexpression causes significantly more apoptosis and lowered proliferation of cancer cells. ZnRF3 overexpression led to greatly reduced levels of Lgr5, a component of Wnt signaling and also Gli1, a component of Hedgehog signaling. Thus, ZnRF3 negatively influences both the Wnt and Hedgehog proliferative pathways, and probably this way it negatively regulates cancer progression. These results suggest the importance of normal ZnRF3 function in checking the progression of cancer cell growth and indicate that a lack of this protein can lead to poorer clinical outcomes for gastric cancer patients. PMID:27352324

  15. 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

  16. Rho kinase acts as a downstream molecule to participate in protein kinase Cε regulation of vascular reactivity after hemorrhagic shock in rats.

    PubMed

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

    2014-09-01

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

  17. OTULIN Antagonizes LUBAC Signaling by Specifically Hydrolyzing Met1-Linked Polyubiquitin

    PubMed Central

    Keusekotten, Kirstin; Elliott, Paul Ronald; Glockner, Laura; Fiil, Berthe Katrine; Damgaard, Rune Busk; Kulathu, Yogesh; Wauer, Tobias; Hospenthal, Manuela Kathrin; Gyrd-Hansen, Mads; Krappmann, Daniel; Hofmann, Kay; Komander, David

    2013-01-01

    Summary The linear ubiquitin (Ub) chain assembly complex (LUBAC) is an E3 ligase that specifically assembles Met1-linked (also known as linear) Ub chains that regulate nuclear factor κB (NF-κB) signaling. Deubiquitinases (DUBs) are key regulators of Ub signaling, but a dedicated DUB for Met1 linkages has not been identified. Here, we reveal a previously unannotated human DUB, OTULIN (also known as FAM105B), which is exquisitely specific for Met1 linkages. Crystal structures of the OTULIN catalytic domain in complex with diubiquitin reveal Met1-specific Ub-binding sites and a mechanism of substrate-assisted catalysis in which the proximal Ub activates the catalytic triad of the protease. Mutation of Ub Glu16 inhibits OTULIN activity by reducing kcat 240-fold. OTULIN overexpression or knockdown affects NF-κB responses to LUBAC, TNFα, and poly(I:C) and sensitizes cells to TNFα-induced cell death. We show that OTULIN binds LUBAC and that overexpression of OTULIN prevents TNFα-induced NEMO association with ubiquitinated RIPK1. Our data suggest that OTULIN regulates Met1-polyUb signaling. PMID:23746843

  18. 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.

  19. 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

  20. Tie2 and Eph Receptor Tyrosine Kinase Activation and Signaling

    PubMed Central

    Barton, William A.; Dalton, Annamarie C.; Seegar, Tom C.M.; Himanen, Juha P.

    2014-01-01

    The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition. PMID:24478383

  1. 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.

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

    PubMed Central

    WU, YAN-HUI; AI, XI; LIU, FU-YAO; LIANG, HUI-FANG; ZHANG, BI-XIANG; CHEN, XIAO-PING

    2016-01-01

    Transforming growth factor (TGF)-β induces cell growth arrest in well-differentiated hepatocellular carcinoma (HCC) while hepatitis B virus X protein (HBx) minimizes the tumor suppression of TGF-β signaling in early chronic hepatitis B. However, how to reverse the oncogenic effect of HBx and sustain the tumor-suppressive action of TGF-β has yet to be investigated. The present study examined the effect of TGF-β and a c-Jun N-terminal kinase (JNK) inhibitor on cell growth in HCC cells with forced expression of HBx. It was found that HBx promoted cell growth via activation of the JNK/pSMAD3L pathway and inhibition of the transforming growth factor-beta type I receptor (TβRI)/pSMAD3C pathway. pSMAD3L/SMAD4 and pSMAD3C/SMAD4 complexes antagonized each other to regulate c-Myc expression. In the absence of HBx, TGF-β induced cell growth arrest through activation of the TβRI/pSMAD3C pathway in well-differentiated HCC cells. In the presence of HBx, TGF-β had no effect on cell growth. JNK inhibitor SP600125 significantly reversed the oncogenic action of HBx and favored TGF-β to regain the ability to inhibit the cell growth in HBx-expressing well-differentiated HCC cells. In conclusion, targeting JNK signaling favors TGF-β to block HBx-induced cell growth promotion in well-differentiated HCC cells. As an adjunct to anti-viral therapy, the combination of TGF-β and inhibition of JNK signaling is a potential therapy for HBV-infected HCC. PMID:26648552

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

    PubMed

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

    2016-02-01

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

  4. H2S- and NO-Signaling Pathways in Alzheimer's Amyloid Vasculopathy: Synergism or Antagonism?

    PubMed Central

    Salmina, Alla B.; Komleva, Yulia K.; Szijártó, István A.; Gorina, Yana V.; Lopatina, Olga L.; Gertsog, Galina E.; Filipovic, Milos R.; Gollasch, Maik

    2015-01-01

    Alzheimer's type of neurodegeneration dramatically affects H2S and NO synthesis and interactions in the brain, which results in dysregulated vasomotor function, brain tissue hypoperfusion and hypoxia, development of perivascular inflammation, promotion of Aβ deposition, and impairment of neurogenesis/angiogenesis. H2S- and NO-signaling pathways have been described to offer protection against Alzheimer's amyloid vasculopathy and neurodegeneration. This review describes recent developments of the increasing relevance of H2S and NO in Alzheimer's disease (AD). More studies are however needed to fully determine their potential use as therapeutic targets in Alzheimer's and other forms of vascular dementia. PMID:26696896

  5. Increasing Notch signaling antagonizes PRC2-mediated silencing to promote reprograming of germ cells into neurons

    PubMed Central

    Seelk, Stefanie; Adrian-Kalchhauser, Irene; Hargitai, Balázs; Hajduskova, Martina; Gutnik, Silvia; Tursun, Baris; Ciosk, Rafal

    2016-01-01

    Cell-fate reprograming is at the heart of development, yet very little is known about the molecular mechanisms promoting or inhibiting reprograming in intact organisms. In the C. elegans germline, reprograming germ cells into somatic cells requires chromatin perturbation. Here, we describe that such reprograming is facilitated by GLP-1/Notch signaling pathway. This is surprising, since this pathway is best known for maintaining undifferentiated germline stem cells/progenitors. Through a combination of genetics, tissue-specific transcriptome analysis, and functional studies of candidate genes, we uncovered a possible explanation for this unexpected role of GLP-1/Notch. We propose that GLP-1/Notch promotes reprograming by activating specific genes, silenced by the Polycomb repressive complex 2 (PRC2), and identify the conserved histone demethylase UTX-1 as a crucial GLP-1/Notch target facilitating reprograming. These findings have wide implications, ranging from development to diseases associated with abnormal Notch signaling. DOI: http://dx.doi.org/10.7554/eLife.15477.001 PMID:27602485

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

    PubMed

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

    2011-10-25

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

  7. 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

  8. 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.

  9. Spatiotemporal brassinosteroid signaling and antagonism with auxin pattern stem cell dynamics in Arabidopsis roots.

    PubMed

    Chaiwanon, Juthamas; Wang, Zhi-Yong

    2015-04-20

    The spatiotemporal balance between stem cell maintenance, proliferation, and differentiation determines the rate of root growth and is controlled by hormones, including auxin and brassinosteroid (BR). However, the spatial actions of BR and its interactions with auxin remain unclear in roots. Here, we show that oppositely patterned and antagonistic actions of BR and auxin maintain the stem cell balance and optimal root growth. We discover a pattern of low levels of nuclear-localized BR-activated transcription factor BZR1 in the stem cell niche and high BZR1 levels in the transition-elongation zone. This BZR1 pattern requires local BR catabolism and auxin synthesis, as well as BR signaling. Cell-type-specific expression of a constitutively active form of BZR1 confirms that the high and low levels of BZR1 are required for the normal cell behaviors in the elongation zone and quiescent center (QC), respectively. Comparison between BR-responsive, BZR1-targeted, auxin-responsive, and developmental zone-specific transcriptomes indicates that BZR1 mostly activates its target genes expressed in the transition-elongation zone, but represses genes in the QC and surrounding stem cells, and that BR and auxin have overall opposite effects on gene expression. Genetic and physiological interactions support that a balance between the antagonistic actions of BR and auxin is required for optimal root growth. These results demonstrate that the level and output specificity of BR signaling are spatially patterned and that, in contrast to their synergism in shoots, BR and auxin interact antagonistically in roots to control the spatiotemporal balance of stem cell dynamics required for optimal root growth.

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

    PubMed

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

    2012-11-28

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

  11. PDK2: the missing piece in the receptor tyrosine kinase signaling pathway puzzle.

    PubMed

    Dong, Lily Q; Liu, Feng

    2005-08-01

    Activation of members of the protein kinase AGC (cAMP dependent, cGMP dependent, and protein kinase C) family is regulated primarily by phosphorylation at two sites: a conserved threonine residue in the activation loop and a serine/threonine residue in a hydrophobic motif (HM) near the COOH terminus. Although phosphorylation of these kinases in the activation loop has been found to be mediated by phosphoinositide-dependent protein kinase-1 (PDK1), the kinase(s) that catalyzes AGC kinase phosphorylation in the HM remains uncharacterized. So far, at least 10 kinases have been suggested to function as an HM kinase or the so-called "PDK2," including mitogen-activated protein (MAP) kinase-activated protein kinase-2 (MK2), integrin-linked kinase (ILK), p38 MAP kinase, protein kinase Calpha (PKCalpha), PKCbeta, the NIMA-related kinase-6 (NEK6), the mammalian target of rapamycin (mTOR), the double-stranded DNA-dependent protein kinase (DNK-PK), and the ataxia telangiectasia mutated (ATM) gene product. However, whether any or all of these kinases act as a physiological HM kinase remains to be established. Nonetheless, available data suggest that multiple systems may be used in cells to regulate the activation of the AGC family kinases. It is possible that, unlike activation loop phosphorylation, phosphorylation of the HM site in the different AGC family kinases is mediated by distinct kinases. In addition, phosphorylation of the AGC family kinase at the HM site could be cell type, signaling pathway, and substrate specific. Identification and characterization of the bonafide HM kinase(s) will be essential to verify these hypotheses. PMID:16014356

  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. Role of SRC family kinases in prolactin signaling.

    PubMed

    Martín-Pérez, Jorge; García-Martínez, José Manuel; Sánchez-Bailón, María Pilar; Mayoral-Varo, Víctor; Calcabrini, Annarica

    2015-01-01

    Prolactin (PRL) is a polypeptide hormone/cytokine mainly synthesized by the lactotrophic cells of the adenohypophysis. In addition to the best-known role in mammary gland development and the functional differentiation of its epithelium, PRL is involved in regulation of multiple physiological processes in higher organisms contributing to their homeostasis. PRL has been also associated with pathology, including breast cancer. Therefore, it is relevant to determine the molecular mechanisms by which PRL controls cellular functions. Here, we analyze the role of Src family kinases (SFKs) in the intracellular signaling pathways controlled by PRL in several model systems. The data show that SFKs are essential components in transmitting signals upon PRL receptor stimulation, as they control activation of Jak2/Stat5 and other routes that regulate PRL cellular responses.

  15. Phosphoinositide 3-kinase mediated signaling in lobster olfactory receptor neurons.

    PubMed

    Corey, Elizabeth A; Bobkov, Yuriy; Pezier, Adeline; Ache, Barry W

    2010-04-01

    In vertebrates and some invertebrates, odorant molecules bind to G protein-coupled receptors on olfactory receptor neurons (ORNs) to initiate signal transduction. Phosphoinositide 3-kinase (PI3K) activity has been implicated physiologically in olfactory signal transduction, suggesting a potential role for a G protein-coupled receptor-activated class I PI3K. Using isoform-specific antibodies, we identified a protein in the olfactory signal transduction compartment of lobster ORNs that is antigenically similar to mammalian PI3Kgamma and cloned a gene for a PI3K with amino acid homology with PI3Kbeta. The lobster olfactory PI3K co-immunoprecipitates with the G protein alpha and beta subunits, and an odorant-evoked increase in phosphatidylinositol (3,4,5)-trisphosphate can be detected in the signal transduction compartment of the ORNs. PI3Kgamma and beta isoform-specific inhibitors reduce the odorant-evoked output of lobster ORNs in vivo. Collectively, these findings provide evidence that PI3K is indeed activated by odorant receptors in lobster ORNs and further support the potential involvement of G protein activated PI3K signaling in olfactory transduction.

  16. 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

  17. Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein tyrosine phosphorylation mediates signal transduction of cellular processes, with protein tyrosine kinases (PTKs) regulating virtually all signaling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into ce...

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

    PubMed

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

    2011-08-01

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

  19. Antagonism of Stem Cell Factor/c-kit Signaling Attenuates Neonatal Chronic Hypoxia-Induced Pulmonary Vascular Remodeling

    PubMed Central

    Young, Karen C; Torres, Eneida; Hehre, Dorothy; Wu, Shu; Suguihara, Cleide; Hare, Joshua M.

    2015-01-01

    Background Accumulating evidence suggests that c-kit positive cells are present in the remodeled pulmonary vasculature bed of patients with pulmonary hypertension (PH). Whether stem cell factor (SCF)/ c-kit regulated pathways potentiate pulmonary vascular remodeling is unknown. Here, we tested the hypothesis that attenuated c-kit signaling would decrease chronic hypoxia-induced pulmonary vascular remodeling by decreasing pulmonary vascular cell mitogenesis. Methods Neonatal FVB/NJ mice treated with non-immune IgG (PL), or c-kit neutralizing antibody (ACK2) as well as c-kit mutant mice (WBB6F1- Kit W− v/ +) and their congenic controls, were exposed to normoxia (FiO2=0.21) or hypoxia (FiO2=0.12) for two weeks. Following this exposure, right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH), pulmonary vascular cell proliferation and remodeling were evaluated. Results As compared to chronically hypoxic controls, c-kit mutant mice had decreased RVSP, RVH, pulmonary vascular remodeling and proliferation. Consistent with these findings, administration of ACK2 to neonatal mice with chronic hypoxia-induced PH decreased RVSP, RVH, pulmonary vascular cell proliferation and remodeling. This attenuation in PH was accompanied by decreased extracellular signal-regulated protein kinase (ERK) 1/2 activation. Conclusion SCF/c-kit signaling may potentiate chronic hypoxia-induced vascular remodeling by modulating ERK activation. Inhibition of c-kit activity may be a potential strategy to alleviate PH. PMID:26705118

  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 2 activates extracellular signal-regulated protein kinase 2

    PubMed Central

    1993-01-01

    Interleukin 2 (IL-2) stimulated activation of the 42-kD extracellular signal-regulated kinase 2 (Erk2) in murine IL-3-dependent cells, expressing either high or intermediate affinity IL-2 receptors. Activation was both rapid, occurring within 5 min of IL-2 addition, and prolonged, remaining elevated for 30 min. Activation of Erk2 appeared to be necessary for IL-2 stimulation of proliferation, as deletion of a region of the cytoplasmic domain of the IL-2 receptor beta chain, essential for IL-2 stimulation of proliferation, abolished Erk2 activation by IL-2. Furthermore, cells that had been deprived of cytokine for 24 h were then refractory to IL-2 stimulation of both Erk2 activity and proliferation. However, elevation of Erk2 activity was not sufficient to stimulate proliferation, as protein kinase C activation stimulated Erk2 activity but not DNA synthesis. Also, cells exposed to IL-2 in the presence of rapamycin showed full Erk2 activation but not DNA synthesis. These data suggest that IL-2 must stimulate both Erk2 activity and a further pathway(s) to trigger cell proliferation. PMID:8376945

  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. Distinct roles for extracellular-signal-regulated protein kinase (ERK) mitogen-activated protein kinases and phosphatidylinositol 3-kinase in the regulation of Mcl-1 synthesis.

    PubMed Central

    Schubert, K M; Duronio, V

    2001-01-01

    Alterations in the expression of various Bcl-2 family members may act as one means by which a cell's survival may be regulated. The mechanism by which cytokines regulate expression of Bcl-2 family members was examined in the haemopoietic cell line TF-1. Cytokine-induced Mcl-1 protein expression was shown to be controlled through a pathway dependent upon phosphatidylinositol 3-kinase (PI 3-kinase). The cytokine-induced increase in mRNA transcription was not dependent upon PI 3-kinase, thus dissociating the immediate-early transcription factors responsible for Mcl-1 transcription from the PI 3-kinase signalling pathway. In contrast, Mcl-1 mRNA levels were dependent upon MEK [mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated protein kinase kinase] activation, suggesting a role for the Ras/MEK/MAPK pathway in Mcl-1 transcription. Activation of PI 3-kinase was shown to be necessary to stimulate Mcl-1 protein translation. This was not due to any effect on prolonging the half-life of the protein. Finally, the lipid second messenger ceramide was shown to cause a reduction in Mcl-1 protein translation, probably via its ability to inhibit protein kinase B activation, providing further clues regarding the death-inducing effect of this lipid. PMID:11368774

  5. Smad2 transduces common signals from receptor serine-threonine and tyrosine kinases.

    PubMed

    de Caestecker, M P; Parks, W T; Frank, C J; Castagnino, P; Bottaro, D P; Roberts, A B; Lechleider, R J

    1998-06-01

    SMAD proteins mediate signals from receptor serine-threonine kinases (RSKs) of the TGF-beta superfamily. We demonstrate here that HGF and EGF, which signal through RTKs, can also mediate SMAD-dependent reporter gene activation and induce rapid phosphorylation of endogenous SMAD proteins by kinase(s) downstream of MEK1. HGF induces phosphorylation and nuclear translocation of epitope-tagged Smad2 and a mutation that blocks TGF-beta signaling also blocks HGF signal transduction. Smad2 may thus act as a common positive effector of TGF-beta- and HGF-induced signals and serve to modulate cross talk between RTK and RSK signaling pathways.

  6. 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

  7. 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

  8. 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

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

    PubMed Central

    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

  10. 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.

  11. FGFR1 signaling stimulates proliferation of human mesenchymal stem cells by inhibiting the cyclin-dependent kinase inhibitors p21(Waf1) and p27(Kip1).

    PubMed

    Dombrowski, Christian; Helledie, Torben; Ling, Ling; Grünert, Martin; Canning, Claire A; Jones, C Michael; Hui, James H; Nurcombe, Victor; van Wijnen, Andre J; Cool, Simon M

    2013-12-01

    Signaling through fibroblast growth factor receptor one (FGFR1) is a known inducer of proliferation in both embryonic and human adult mesenchymal stem cells (hMSCs) and positively regulates maintenance of stem cell viability. Leveraging the mitogenic potential of FGF2/FGFR1 signaling in stem cells for therapeutic applications necessitates a mechanistic understanding of how this receptor stimulates cell cycle progression. Using small interfering RNA (siRNA) depletion, antibody-inhibition, and small molecule inhibition, we establish that FGFR1 activity is rate limiting for self-renewal of hMSCs. We show that FGFR1 promotes stem cell proliferation through multiple mechanisms that unite to antagonize cyclin-dependent kinase (CDK) inhibitors. FGFR1 not only stimulates c-Myc to suppress transcription of the CDK inhibitors p21(Waf1) and p27(Kip1), thus promoting cell cycle progression but also increases the activity of protein kinase B (AKT) and the level of S-phase kinase-associated protein 2 (Skp2), resulting in the nuclear exclusion and reduction of p21(Waf1). The in vivo importance of FGFR1 signaling for the control of proliferation in mesenchymal progenitor populations is underscored by defects in ventral mesoderm formation during development upon inhibition of its signaling. Collectively, these studies demonstrate that FGFR1 signaling mediates the continuation of MSC growth and establishes a receptor target for enhancing the expansion of mesenchymal progenitors while maintaining their multilineage potential.

  12. AGCVIII Kinases: at the crossroads of cellular signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    AGCVIII kinases regulate diverse developmental and cellular processes in plants. As putative mediators of secondary messengers, AGCVIII kinases potentially integrate developmental and environmental cues into specific cellular responses through substrate phosphorylation. Here we discuss the functiona...

  13. The role of the extracellular signal-regulated kinase signaling pathway in mood modulation.

    PubMed

    Einat, Haim; Yuan, Peixiong; Gould, Todd D; Li, Jianling; Du, JianHua; Zhang, Lei; Manji, Husseini K; Chen, Guang

    2003-08-13

    The neurobiological underpinnings of mood modulation, molecular pathophysiology of manic-depressive illness, and therapeutic mechanism of mood stabilizers are largely unknown. The extracellular signal-regulated kinase (ERK) pathway is activated by neurotrophins and other neuroactive chemicals to produce their effects on neuronal differentiation, survival, regeneration, and structural and functional plasticity. We found that lithium and valproate, commonly used mood stabilizers for the treatment of manic-depressive illness, stimulated the ERK pathway in the rat hippocampus and frontal cortex. Both drugs increased the levels of activated phospho-ERK44/42, activated phospho-ribosomal protein S6 kinase-1 (RSK1) (a substrate of ERK), phospho-CREB (cAMP response element-binding protein) and phospho-B cell lymphoma protein-2 antagonist of cell death (substrates of RSK), and BDNF. Inhibiting the ERK pathway with the blood-brain barrier-penetrating mitogen-activated protein kinase (MAP kinase)/ERK kinase (MEK) kinase inhibitor SL327, but not with the nonblood-brain barrier-penetrating MEK inhibitor U0126, decreased immobility time and increased swimming time of rats in the forced-swim test. SL327, but not U0126, also increased locomotion time and distance traveled in a large open field. The behavioral changes in the open field were prevented with chronic lithium pretreatment. SL327-induced behavioral changes are qualitatively similar to the changes induced by amphetamine, a compound that induces relapse in remitted manic patients and mood elevation in normal subjects. These data suggest that the ERK pathway may mediate the antimanic effects of mood stabilizers.

  14. 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

  15. Parallel regulation of mitogen-activated protein kinase kinase 3 (MKK3) and MKK6 in Gq-signaling cascade.

    PubMed

    Yamauchi, J; Tsujimoto, G; Kaziro, Y; Itoh, H

    2001-06-29

    Heterotrimeric G protein G(q) stimulates the activity of p38 mitogen-activated protein kinase (MAPK) in mammalian cells. To investigate the signaling mechanism whereby alpha and betagamma subunits of G(q) activate p38 MAPK, we introduced kinase-deficient mutants of mitogen-activated protein kinase kinase 3 (MKK3), MKK4, and MKK6 into human embryonal kidney 293 cells. The activation of p38 MAPK by Galpha(q) and Gbetagamma was blocked by kinase-deficient MKK3 and MKK6 but not by kinase-deficient MKK4. In addition, Galpha(q) and Gbetagamma stimulated MKK3 and MKK6 activities. The MKK3 and MKK6 activations by Galpha(q), but not by Gbetagamma, were dependent on phospholipase C and c-Src. Galpha(q) stimulated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-dependent manner. On the other hand, Gbetagamma activated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-, Rac-, and Cdc42-dependent manner. Gbetagamma-induced MKK3 and MKK6 activations were dependent on a tyrosine kinase other than c-Src. These results suggest that Galpha(q) and Gbetagamma stimulate the activity of p38 MAPK by regulating MKK3 and MKK6 through parallel signaling pathways.

  16. Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation.

    PubMed Central

    Weng, Q P; Andrabi, K; Klippel, A; Kozlowski, M T; Williams, L T; Avruch, J

    1995-01-01

    The p70 S6 kinase is activated by insulin and mitogens through multisite phosphorylation of the enzyme. One set of activating phosphorylations occurs in a putative autoinhibitory domain in the noncatalytic carboxyl-terminal tail. Deletion of this tail yields a variant (p70 delta CT104) that nevertheless continues to be mitogen regulated. Coexpression with a recombinant constitutively active phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) gives substantial activation of both full-length p70 and p70 delta CT104 but not Rsk. Activation of p70 delta CT104 by PI 3-kinase and inhibition by wortmannin are each accompanied by parallel and selective changes in the phosphorylation of p70 Thr-252. A Thr or Ser at this site, in subdomain VIII of the catalytic domain just amino-terminal to the APE motif, is necessary for p70 40S kinase activity. The inactive ATP-binding site mutant K123M p70 delta CT104 undergoes phosphorylation of Thr-252 in situ but does not undergo direct phosphorylation by the active PI 3-kinase in vitro. PI 3-kinase provides a signal necessary for the mitogen activation of the p70 S6 kinase, which directs the site-specific phosphorylation of Thr-252 in the p70 catalytic domain, through a distinctive signal transduction pathway. Images Fig. 1 Fig. 2 Fig. 3 PMID:7777579

  17. c-Jun controls the efficiency of MAP kinase signaling by transcriptional repression of MAP kinase phosphatases

    SciTech Connect

    Sprowles, Amy; Wu Yimi; Kung, H.-J.; Wisdom, Ron . E-mail: ronald.wisdom@ucdmc.ucdavis.edu

    2005-08-15

    The mammalian JNK signaling pathway regulates the transcriptional response of cells to environmental stress, including UV irradiation. This signaling pathway is composed of a classical MAP kinase cascade; activation results in phosphorylation of the transcription factor substrates c-Jun and ATF2, and leads to changes in gene expression. The defining components of this pathway are conserved in the fission yeast S. pombe, where the genetic studies have shown that the ability of the JNK homolog Spc1 to be activated in response to UV irradiation is dependent on the presence of the transcription factor substrate Atf1. We have used genetic analysis to define the role of c-Jun in activation of the mammalian JNK signaling pathway. Our results show that optimal activation of JNK requires the presence of its transcription factor substrate c-Jun. Mutational analysis shows that the ability of c-Jun to support efficient activation of JNK requires the ability of Jun to bind DNA, suggesting a transcriptional mechanism. Consistent with this, we show that c-Jun represses the expression of several MAP kinase phosphatases. In the absence of c-Jun, the increased expression of MAP kinase phosphatases leads to impaired activation of the ERK, JNK, and p38 MAP kinases after pathway activation. The results show that one function of c-Jun is to regulate the efficiency of signaling by the ERK, p38, and JNK MAP kinases, a function that is likely to affect cellular responses to many different stimuli.

  18. 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

  19. Jun N-terminal kinase signaling makes a face

    PubMed Central

    Hursh, Deborah A.; Stultz, Brian G.; Park, Sung Yeon

    2016-01-01

    ABSTRACT decapentaplegic (dpp), the Drosophila ortholog of BMP 2/4, directs ventral adult head morphogenesis through expression in the peripodial epithelium of the eye-antennal disc. This dpp expressing domain exerts effects both on the peripodial epithelium, and the underlying disc proper epithelium. We have uncovered a role for the Jun N-terminal kinase (JNK) pathway in dpp-mediated ventral head development. JNK activity is required for dpp's action on the disc proper, but in the absence of dpp expression, excessive JNK activity is produced, leading to specific loss of maxillary palps. In this review we outline our hypotheses on how dpp acts by both short range and longer range mechanisms to direct head morphogenesis and speculate on the dual role of JNK signaling in this process. Finally, we describe the regulatory control of dpp expression in the eye-antennal disc, and pose the problem of how the various expression domains of a secreted protein can be targeted to their specific functions. PMID:27384866

  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.

  1. Jun N-terminal kinase signaling makes a face.

    PubMed

    Hursh, Deborah A; Stultz, Brian G; Park, Sung Yeon

    2016-10-01

    decapentaplegic (dpp), the Drosophila ortholog of BMP 2/4, directs ventral adult head morphogenesis through expression in the peripodial epithelium of the eye-antennal disc. This dpp expressing domain exerts effects both on the peripodial epithelium, and the underlying disc proper epithelium. We have uncovered a role for the Jun N-terminal kinase (JNK) pathway in dpp-mediated ventral head development. JNK activity is required for dpp's action on the disc proper, but in the absence of dpp expression, excessive JNK activity is produced, leading to specific loss of maxillary palps. In this review we outline our hypotheses on how dpp acts by both short range and longer range mechanisms to direct head morphogenesis and speculate on the dual role of JNK signaling in this process. Finally, we describe the regulatory control of dpp expression in the eye-antennal disc, and pose the problem of how the various expression domains of a secreted protein can be targeted to their specific functions.

  2. 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

  3. TGF-β activates Erk MAP kinase signalling through direct phosphorylation of ShcA

    PubMed Central

    Lee, Matt K; Pardoux, Cécile; Hall, Marie C; Lee, Pierre S; Warburton, David; Qing, Jing; Smith, Susan M; Derynck, Rik

    2007-01-01

    Erk1/Erk2 MAP kinases are key regulators of cell behaviour and their activation is generally associated with tyrosine kinase signalling. However, TGF-β stimulation also activates Erk MAP kinases through an undefined mechanism, albeit to a much lower level than receptor tyrosine kinase stimulation. We report that upon TGF-β stimulation, the activated TGF-β type I receptor (TβRI) recruits and directly phosphorylates ShcA proteins on tyrosine and serine. This dual phosphorylation results from an intrinsic TβRI tyrosine kinase activity that complements its well-defined serine-threonine kinase function. TGF-β-induced ShcA phosphorylation induces ShcA association with Grb2 and Sos, thereby initiating the well-characterised pathway linking receptor tyrosine kinases with Erk MAP kinases. We also found that TβRI is tyrosine phosphorylated in response to TGF-β. Thus, TβRI, like the TGF-β type II receptor, is a dual-specificity kinase. Recruitment of tyrosine kinase signalling pathways may account for aspects of TGF-β biology that are independent of Smad signalling. PMID:17673906

  4. dpl-1 DP and efl-1 E2F act with lin-35 Rb to antagonize Ras signaling in C. elegans vulval development.

    PubMed

    Ceol, C J; Horvitz, H R

    2001-03-01

    The synthetic multivulva (synMuv) genes define two functionally redundant pathways that antagonize RTK/Ras signaling during Caenorhabditis elegans vulval induction. The synMuv gene lin-35 encodes a protein similar to the mammalian tumor suppressor pRB and has been proposed to act as a transcriptional repressor. Studies using mammalian cells have shown that pRB can prevent cell cycle progression by inhibiting DP/E2F-mediated transcriptional activation. We identified C. elegans genes that encode proteins similar to DP or E2F. Loss-of-function mutations in two of these genes, dpl-1 DP and efl-1 E2F, caused the same vulval abnormalities as do lin-35 Rb loss-of-function mutations. We propose that rather than being inhibited by lin-35 Rb, dpl-1 DP and efl-1 E2F act with lin-35 Rb in transcriptional repression to antagonize RTK/Ras signaling during vulval development.

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

    PubMed

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

    1999-01-22

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

  6. Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-releasing Hormone*

    PubMed Central

    Higa-Nakamine, Sayomi; Maeda, Noriko; Toku, Seikichi; Yamamoto, Hideyuki

    2015-01-01

    The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1–7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of Gq/11 proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer. PMID:26338704

  7. 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

  8. Manipulation of Mitogen-Activated Protein Kinase Kinase Signaling in the Arabidopsis Stomatal Lineage Reveals Motifs That Contribute to Protein Localization and Signaling Specificity[W][OPEN

    PubMed Central

    Lampard, Gregory R.; Wengier, Diego L.; Bergmann, Dominique C.

    2014-01-01

    When multiple mitogen-activated protein kinase (MAPK) components are recruited recurrently to transduce signals of different origins, and often opposing outcomes, mechanisms to enforce signaling specificity are of utmost importance. These mechanisms are largely uncharacterized in plant MAPK signaling networks. The Arabidopsis thaliana stomatal lineage was previously used to show that when rendered constitutively active, four MAPK kinases (MKKs), MKK4/5/7/9, are capable of perturbing stomatal development and that these kinases comprise two pairs, MKK4/5 and MKK7/9, with both overlapping and divergent functions. We characterized the contributions of specific structural domains of these four “stomatal” MKKs to MAPK signaling output and specificity both in vitro and in vivo within the three discrete cell types of the stomatal lineage. These results verify the influence of functional docking (D) domains of MKKs on MAPK signal output and identify novel regulatory functions for previously uncharacterized structures within the N termini of MKK4/5. Beyond this, we present a novel function of the D-domains of MKK7/9 in regulating the subcellular localization of these kinases. These results provide tools to broadly assess the extent to which these and additional motifs within MKKs function to regulate MAPK signal output throughout the plant. PMID:25172143

  9. Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase.

    PubMed

    Venkitachalam, Srividya; Chueh, Fu-Yu; Leong, King-Fu; Pabich, Samantha; Yu, Chao-Lan

    2011-03-01

    Lymphocyte-specific protein tyrosine kinase (Lck) plays a key role in T cell signal transduction and is tightly regulated by phosphorylation and dephosphorylation. Lck can function as an oncoprotein when overexpressed or constantly activated by mutations. Our previous studies showed that Lck-induced cellular transformation could be suppressed by enforced expression of suppressor of cytokine signaling 1 (SOCS1), a SOCS family member involved in the negative feedback control of cytokine signaling. We observed attenuated Lck kinase activity in SOCS1-expressing cells, suggesting an important role of SOCS in regulating Lck functions. It remains largely unknown whether and how SOCS proteins interact with the oncogenic Lck kinase. Here, we report that among four SOCS family proteins, SOCS1, SOCS2, SOCS3 and CIS (cytokine-inducible SH2 domain containing protein), SOCS1 has the highest affinity in binding to the oncogenic Lck kinase. We identified the positive regulatory phosphotyrosine 394 residue in the kinase domain as the key interacting determinant in Lck. Additionally, the Lck kinase domain alone is sufficient to bind SOCS1. While the SH2 domain in SOCS1 is important in its association with the oncogenic Lck kinase, other functional domains may also contribute to overall binding affinity. These findings provide important mechanistic insights into the role of SOCS proteins as tumor suppressors in cells transformed by oncogenic protein tyrosine kinases.

  10. Intracellular Signaling by Hydrolysis of Phospholipids and Activation of Protein Kinase C

    NASA Astrophysics Data System (ADS)

    Nishizuka, Yasutomi

    1992-10-01

    Hydrolysis of inositol phospholipids by phospholipase C is initiated by either receptor stimulation or opening of Ca2+ channels. This was once thought to be the sole mechanism to produce the diacylglycerol that links extracellular signals to intracellular events through activation of protein kinase C. It is becoming clear that agonist-induced hydrolysis of other membrane phospholipids, particularly choline phospholipids, by phospholipase D and phospholipase A_2 may also take part in cell signaling. The products of hydrolysis of these phospholipids may enhance and prolong the activation of protein kinase C. Such prolonged activation of protein kinase C is essential for long-term cellular responses such as cell proliferation and differentiation.

  11. The role of protein kinase C in cell surface signal transduction and tumour promotion

    NASA Astrophysics Data System (ADS)

    Nishizuka, Yasutomi

    1984-04-01

    Protein kinase C has a crucial role in signal transduction for a variety of biologically active substances which activate cellular functions and proliferation. When cells are stimulated, protein kinase C is transiently activated by diacylglycerol which is produced in the membrane during the signal-induced turnover of inositol phospholipids. Tumour-promoting phorbol esters, when intercalated into the cell membrane, may substitute for diacylglycerol and permanently activate protein kinase C. The enzyme probably serves as a receptor for the tumour promoters. Further exploration of the roles of this enzyme may provide clues for understanding the mechanism of cell growth and differentiation.

  12. 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

  13. Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase

    PubMed Central

    Jiang, Youwei; Cypess, Aaron M.; Muse, Evan D.; Wu, Cui-Rong; Unson, Cecilia G.; Merrifield, R. B.; Sakmar, Thomas P.

    2001-01-01

    We prepared a stable cell line expressing the glucagon receptor to characterize the effect of Gs-coupled receptor stimulation on extracellular signal-regulated protein kinase 1/2 (ERK1/2) activity. Glucagon treatment of the cell line caused a dose-dependent increase in cAMP concentration, activation of cAMP-dependent protein kinase (PKA), and transient release of intracellular calcium. Glucagon treatment also caused rapid dose-dependent phosphorylation and activation of mitogen-activated protein kinase kinase/ERK kinase (MEK1/2) and ERK1/2. Inhibition of either PKA or MEK1/2 blocked ERK1/2 activation by glucagon. However, no significant activation of several upstream activators of MEK, including Ras, Rap1, and Raf, was observed in response to glucagon treatment. In addition, chelation of intracellular calcium reduced glucagon-mediated ERK1/2 activation. In transient transfection experiments, glucagon receptor mutants that bound glucagon but failed to increase intracellular cAMP and calcium concentrations showed no glucagon-stimulated ERK1/2 phosphorylation. We conclude that glucagon-induced MEK1/2 and ERK1/2 activation is mediated by PKA and that an increase in intracellular calcium concentration is required for maximal ERK activation. PMID:11517300

  14. 5-HT2B antagonism arrests non-canonical TGF-β1-induced valvular myofibroblast differentiation

    PubMed Central

    Hutcheson, Joshua D.; Ryzhova, Larisa M.; Setola, Vincent; Merryman, W. David

    2012-01-01

    Transforming growth factor-β1 (TGF-β1) induces myofibroblast activation of quiescent aortic valve interstitial cells (AVICs), a differentiation process implicated in calcific aortic valve disease (CAVD). The ubiquity of TGF-β1 signaling makes it difficult to target in a tissue specific manner; however, the serotonin 2B receptor (5-HT2B) is highly localized to cardiopulmonary tissues and agonism of this receptor displays pro-fibrotic effects in a TGF-β1-dependent manner. Therefore, we hypothesized that antagonism of 5-HT2B opposes TGF-β1-induced pathologic differentiation of AVICs and may offer a druggable target to prevent CAVD. To test this hypothesis, we assessed the interaction of 5-HT2B antagonism with canonical and non-canonical TGF-β1 pathways to inhibit TGF-β1-induced activation of isolated porcine AVICs in vitro. Here we show that AVIC activation and subsequent calcific nodule formation is completely mitigated by 5-HT2B antagonism. Interestingly, 5-HT2B antagonism does not inhibit canonical TGF-β1 signaling as identified by Smad3 phosphorylation and activation of a partial plasminogen activator inhibitor-1 promoter (PAI-1, a transcriptional target of Smad3), but prevents non-canonical p38 MAPK phosphorylation. It was initially suspected that 5-HT2B antagonism prevents Src tyrosine kinase phosphorylation; however, we found that this is not the case and time-lapse microscopy indicates that 5-HT2B antagonism prevents non-canonical TGF-β1 signaling by physically arresting Src tyrosine kinase. This study demonstrates the necessity of non-canonical TGF-β1 signaling in leading to pathologic AVIC differentiation. Moreover, we believe that the results of this study suggest 5-HT2B antagonism as a novel therapeutic approach for CAVD that merits further investigation. PMID:22940605

  15. 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

  16. 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

  17. Antagonism of notch signaling activity by members of a novel protein family encoded by the bearded and enhancer of split gene complexes.

    PubMed

    Lai, E C; Bodner, R; Kavaler, J; Freschi, G; Posakony, J W

    2000-01-01

    Cell-cell signaling through the Notch receptor is a principal mechanism underlying cell fate specification in a variety of developmental processes in metazoans, such as neurogenesis. In this report we describe our investigation of seven members of a novel gene family in Drosophila with important connections to Notch signaling. These genes all encode small proteins containing predicted basic amphipathic (&agr;)-helical domains in their amino-terminal regions, as described originally for Bearded; accordingly, we refer to them as Bearded family genes. Five members of the Bearded family are located in a newly discovered gene complex, the Bearded Complex; two others reside in the previously identified Enhancer of split Complex. All members of this family contain, in their proximal upstream regions, at least one high-affinity binding site for the Notch-activated transcription factor Suppressor of Hairless, suggesting that all are directly regulated by the Notch pathway. Consistent with this, we show that Bearded family genes are expressed in a variety of territories in imaginal tissue that correspond to sites of active Notch signaling. We demonstrate that overexpression of any family member antagonizes the activity of the Notch pathway in multiple cell fate decisions during adult sensory organ development. These results suggest that Bearded family genes encode a novel class of effectors or modulators of Notch signaling.

  18. 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.

  19. 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

  20. Signal Transduction by BvgS Sensor Kinase

    PubMed Central

    Dupré, Elian; Lesne, Elodie; Guérin, Jérémy; Lensink, Marc F.; Verger, Alexis; de Ruyck, Jérôme; Brysbaert, Guillaume; Vezin, Hervé; Locht, Camille; Antoine, Rudy; Jacob-Dubuisson, Françoise

    2015-01-01

    The two-component sensory transduction system BvgAS controls the virulence regulon of the whooping-cough agent Bordetella pertussis. The periplasmic moiety of the homodimeric sensor kinase BvgS is composed of four bilobed Venus flytrap (VFT) perception domains followed by α helices that extend into the cytoplasmic membrane. In the virulent phase, the default state of B. pertussis, the cytoplasmic enzymatic moiety of BvgS acts as kinase by autophosphorylating and transferring the phosphoryl group to the response regulator BvgA. Under laboratory conditions, BvgS shifts to phosphatase activity in response to modulators, notably nicotinate ions. Here we characterized the effects of nicotinate and related modulators on the BvgS periplasmic moiety by using site-directed mutagenesis and in silico and biophysical approaches. Modulators bind with low affinity to BvgS in the VFT2 cavity. Electron paramagnetic resonance shows that their binding globally affects the conformation and dynamics of the periplasmic moiety. Specific amino acid substitutions designed to slacken interactions within and between the VFT lobes prevent BvgS from responding to nicotinate, showing that BvgS shifts from kinase to phosphatase activity in response to this modulator via a tense transition state that involves a large periplasmic structural block. We propose that this transition enables the transmembrane helices to adopt a distinct conformation that sets the cytoplasmic enzymatic moiety in the phosphatase mode. The bona fide, in vivo VFT ligands that remain to be identified are likely to trigger similar effects on the transmembrane and cytoplasmic moieties. This mechanism may be relevant to the other VFT-containing sensor kinases homologous to BvgS. PMID:26203186

  1. Cholinergic Signaling through Synaptic SK Channels: It’s a Protein Kinase but Which One?

    PubMed Central

    Maylie, James; Adelman, John P.

    2016-01-01

    Cholinergic signaling modulates synaptic responses and influences cognition. In this issue of Neuron, two groups (Buchanan et al. and Giessel and Sabatini) present evidence that cholinergic signaling enhances postsynaptic responses in CA1 neurons by decreasing synaptic SK channel activity. However, they come to different conclusions about the protein kinases involved in this process. PMID:21144995

  2. 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.

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

    PubMed Central

    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

    2013-01-01

    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. PMID:22116026

  4. Spatially coordinated kinase signaling regulates local axon degeneration.

    PubMed

    Chen, Mark; Maloney, Janice A; Kallop, Dara Y; Atwal, Jasvinder K; Tam, Stephen J; Baer, Kristin; Kissel, Holger; Kaminker, Joshua S; Lewcock, Joseph W; Weimer, Robby M; Watts, Ryan J

    2012-09-26

    In addition to being a hallmark of neurodegenerative disease, axon degeneration is used during development of the nervous system to prune unwanted connections. In development, axon degeneration is tightly regulated both temporally and spatially. Here, we provide evidence that degeneration cues are transduced through various kinase pathways functioning in spatially distinct compartments to regulate axon degeneration. Intriguingly, glycogen synthase kinase-3 (GSK3) acts centrally, likely modulating gene expression in the cell body to regulate distally restricted axon degeneration. Through a combination of genetic and pharmacological manipulations, including the generation of an analog-sensitive kinase allele mutant mouse for GSK3β, we show that the β isoform of GSK3, not the α isoform, is essential for developmental axon pruning in vitro and in vivo. Additionally, we identify the dleu2/mir15a/16-1 cluster, previously characterized as a regulator of B-cell proliferation, and the transcription factor tbx6, as likely downstream effectors of GSK3β in axon degeneration.

  5. NMDA receptor subunits and associated signaling molecules mediating antidepressant-related effects of NMDA-GluN2B antagonism

    PubMed Central

    Kiselycznyk, Carly; Jury, Nicholas; Halladay, Lindsay; Nakazawa, Kazu; Mishina, Masayoshi; Sprengel, Rolf; Grant, Seth G.N.; Svenningsson, Per; Holmes, Andrew

    2015-01-01

    Drugs targeting the glutamate N-methyl-D-aspartate receptor (NMDAR) may be efficacious for treating mood disorders, as exemplified by the rapid antidepressant effects produced by single administration of the NMDAR antagonist ketamine. Though the precise mechanisms underlying the antidepressant-related effects of NMDAR antagonism remain unclear, recent studies implicate specific NMDAR subunits, including GluN2A and GluN2B, as well as the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) subunit glutamate receptor interacting molecule, PSD-95. Here, integrating mutant and pharmacological in mice, we investigated the contribution of these subunits and molecules to antidepressant-related behaviors and the antidepressant-related effects of the GluN2B blocker, Ro 25-6981. We found that global deletion of GluA1 or PSD-95 reduced forced swim test (FST) immobility, mimicking the antidepressant-related effect produced by systemically administered Ro 25-6981 in C57BL/6J mice. Moreover, the FST antidepressant-like effects of systemic Ro 25-6981 were intact in mutants with global GluA1 deletion or GluN1 deletion in forebrain interneurons, but were absent in mutants constitutively lacking GluN2A or PSD-95. Next, we found that microinfusing Ro 25-6981 into the medial prefrontal cortex (mPFC), but not basolateral amygdala, of C57BL/6J mice was sufficient to produce an antidepressant-like effect. Together, these findings extend and refine current understanding of the mechanisms mediating antidepressant-like effects produced by NMDAR-GluN2B antagonists, and may inform the development of a novel class of medications for treating depression that target the GluN2B subtype of NMDAR. PMID:25800971

  6. Lithium protection of phencyclidine-induced neurotoxicity in developing brain: the role of phosphatidylinositol-3 kinase/Akt and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathways.

    PubMed

    Xia, Yan; Wang, Cheng Z; Liu, Jie; Anastasio, Noelle C; Johnson, Kenneth M

    2008-09-01

    Phencyclidine (PCP) and other N-methyl-D-aspartate (NMDA) receptor antagonists have been shown to be neurotoxic to developing brains and to result in schizophrenia-like behaviors later in development. Prevention of both effects by antischizophrenic drugs suggests the validity of PCP neurodevelopmental toxicity as a heuristic model of schizophrenia. Lithium is used for the treatment of bipolar and schizoaffective disorders and has recently been shown to have neuroprotective properties. The present study used organotypic corticostriatal slices taken from postnatal day 2 rat pups to investigate the protective effect of lithium and the role of the phosphatidylinositol-3 kinase (PI-3K)/Akt and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathways in PCP-induced cell death. Lithium pretreatment dose-dependently reduced PCP-induced caspase-3 activation and DNA fragmentation in layers II to IV of the cortex. PCP elicited time-dependent inhibition of the MEK/ERK and PI-3K/Akt pathways, as indicated by dephosphorylation of ERK1/2 and Akt. The proapoptotic factor glycogen synthase kinase (GSK)-3beta was also dephosphorylated at serine 9 and thus activated. Lithium prevented PCP-induced inhibition of the two pathways and activation of GSK-3beta. Furthermore, blocking either PI-3K/Akt or MEK/ERK pathway abolished the protective effect of lithium, whereas inhibiting GSK-3beta activity mimicked the protective effect of lithium. However, no cross-talk between the two pathways was found. Finally, specific GSK-3beta inhibition did not prevent PCP-induced dephosphorylation of Akt and ERK. These data strongly suggest that the protective effect of lithium against PCP-induced neuroapoptosis is mediated through independent stimulation of the PI-3K/Akt and ERK pathways and suppression of GSK-3beta activity.

  7. PI3 Kinase signals BCR dependent mature B cell survival

    PubMed Central

    Srinivasan, Lakshmi; Sasaki, Yoshiteru; Calado, Dinis Pedro; Zhang, Baochun; Paik, Ji Hye; DePinho, Ronald A.; Kutok, Jeffrey L.; Kearney, John F.; Otipoby, Kevin L.; Rajewsky, Klaus

    2009-01-01

    Summary Previous work has shown that mature B cells depend upon survival signals delivered to the cells by their antigen receptor (BCR). To identify the molecular nature of this survival signal, we have developed a genetic approach in which ablation of the BCR is combined with the activation of specific, BCR dependent signaling cascades in mature B cells in vivo. Using this system, we provide evidence that the survival of BCR deficient mature B cells can be rescued by a single signaling pathway downstream of the BCR, namely PI3K signaling, with the FOXO1 transcription factor playing a central role. PMID:19879843

  8. The calcineurin signaling network evolves via conserved kinase-phosphatase modules that transcend substrate identity.

    PubMed

    Goldman, Aaron; Roy, Jagoree; Bodenmiller, Bernd; Wanka, Stefanie; Landry, Christian R; Aebersold, Ruedi; Cyert, Martha S

    2014-08-01

    To define a functional network for calcineurin, the conserved Ca(2+)/calmodulin-regulated phosphatase, we systematically identified its substrates in S. cerevisiae using phosphoproteomics and bioinformatics, followed by copurification and dephosphorylation assays. This study establishes new calcineurin functions and reveals mechanisms that shape calcineurin network evolution. Analyses of closely related yeasts show that many proteins were recently recruited to the network by acquiring a calcineurin-recognition motif. Calcineurin substrates in yeast and mammals are distinct due to network rewiring but, surprisingly, are phosphorylated by similar kinases. We postulate that corecognition of conserved substrate features, including phosphorylation and docking motifs, preserves calcineurin-kinase opposition during evolution. One example we document is a composite docking site that confers substrate recognition by both calcineurin and MAPK. We propose that conserved kinase-phosphatase pairs define the architecture of signaling networks and allow other connections between kinases and phosphatases to develop that establish common regulatory motifs in signaling networks.

  9. 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...

  10. 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

  11. 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.

  12. 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

  13. Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Arrestin-Src Kinase Interaction.

    PubMed

    Wagener, Brant M; Marjon, Nicole A; Prossnitz, Eric R

    2016-01-01

    Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking.

  14. Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Arrestin-Src Kinase Interaction

    PubMed Central

    Wagener, Brant M.; Marjon, Nicole A.; Prossnitz, Eric R.

    2016-01-01

    Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking. PMID:26788723

  15. Microbial Degradation of Cellular Kinases Impairs Innate Immune Signaling and Paracrine TNFα Responses

    PubMed Central

    Barth, Kenneth; Genco, Caroline Attardo

    2016-01-01

    The NFκB and MAPK signaling pathways are critical components of innate immunity that orchestrate appropriate immune responses to control and eradicate pathogens. Their activation results in the induction of proinflammatory mediators, such as TNFα a potent bioactive molecule commonly secreted by recruited inflammatory cells, allowing for paracrine signaling at the site of an infection. In this study we identified a novel mechanism by which the opportunistic pathogen Porphyromonas gingivalis dampens innate immune responses by disruption of kinase signaling and degradation of inflammatory mediators. The intracellular immune kinases RIPK1, TAK1, and AKT were selectively degraded by the P. gingivalis lysine-specific gingipain (Kgp) in human endothelial cells, which correlated with dysregulated innate immune signaling. Kgp was also observed to attenuate endothelial responsiveness to TNFα, resulting in a reduction in signal flux through AKT, ERK and NFκB pathways, as well as a decrease in downstream proinflammatory mRNA induction of cytokines, chemokines and adhesion molecules. A deficiency in Kgp activity negated decreases to host cell kinase protein levels and responsiveness to TNFα. Given the essential role of kinase signaling in immune responses, these findings highlight a unique mechanism of pathogen-induced immune dysregulation through inhibition of cell activation, paracrine signaling, and dampened cellular proinflammatory responses. PMID:27698456

  16. 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.

  17. 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

  18. 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

  19. Compartmentalization role of A-kinase anchoring proteins (AKAPs) in mediating protein kinase A (PKA) signaling and cardiomyocyte hypertrophy.

    PubMed

    Rababa'h, Abeer; Singh, Sonal; Suryavanshi, Santosh V; Altarabsheh, Salah Eldien; Deo, Salil V; McConnell, Bradley K

    2014-12-24

    The Beta-adrenergic receptors (β-ARs) stimulation enhances contractility through protein kinase-A (PKA) substrate phosphorylation. This PKA signaling is conferred in part by PKA binding to A-kinase anchoring proteins (AKAPs). AKAPs coordinate multi-protein signaling networks that are targeted to specific intracellular locations, resulting in the localization of enzyme activity and transmitting intracellular actions of neurotransmitters and hormones to its target substrates. In particular, mAKAP (muscle-selective AKAP) has been shown to be present on the nuclear envelope of cardiomyocytes with various proteins including: PKA-regulatory subunit (RIIα), phosphodiesterase-4D3, protein phosphatase-2A, and ryanodine receptor (RyR2). Therefore, through the coordination of spatial-temporal signaling of proteins and enzymes, mAKAP controls cyclic-adenosine monophosphate (cAMP) levels very tightly and functions as a regulator of PKA-mediated substrate phosphorylation leading to changes in calcium availability and myofilament calcium sensitivity. The goal of this review is to elucidate the critical compartmentalization role of mAKAP in mediating PKA signaling and regulating cardiomyocyte hypertrophy by acting as a scaffolding protein. Based on our literature search and studying the structure-function relationship between AKAP scaffolding protein and its binding partners, we propose possible explanations for the mechanism by which mAKAP promotes cardiac hypertrophy.

  20. Protein kinase C in the immune system: from signalling to chromatin regulation.

    PubMed

    Lim, Pek Siew; Sutton, Christopher Ray; Rao, Sudha

    2015-12-01

    Protein kinase C (PKC) form a key family of enzymes involved in signalling pathways that specifically phosphorylates substrates at serine/threonine residues. Phosphorylation by PKC is important in regulating a variety of cellular events such as cell proliferation and the regulation of gene expression. In the immune system, PKCs are involved in regulating signal transduction pathways important for both innate and adaptive immunity, ultimately resulting in the expression of key immune genes. PKCs act as mediators during immune cell signalling through the immunological synapse. PKCs are traditionally known to be cytoplasmic signal transducers and are well embedded in the signalling pathways of cells to mediate the cells' response to a stimulus from the plasma membrane to the nucleus. PKCs are also found to transduce signals within the nucleus, a process that is distinct from the cytoplasmic signalling pathway. There is now growing evidence suggesting that PKC can directly regulate gene expression programmes through a non-traditional role as nuclear kinases. In this review, we will focus on the role of PKCs as key cytoplasmic signal transducers in immune cell signalling, as well as its role in nuclear signal transduction. We will also highlight recent evidence for its newly discovered regulatory role in the nucleus as a chromatin-associated kinase.

  1. R-spondin 3 regulates dorsoventral and anteroposterior patterning by antagonizing Wnt/β-catenin signaling in zebrafish embryos.

    PubMed

    Rong, Xiaozhi; Chen, Chen; Zhou, Pin; Zhou, Yumei; Li, Yun; Lu, Ling; Liu, Yunzhang; Zhou, Jianfeng; Duan, Cunming

    2014-01-01

    The Wnt/β-catenin or canonical Wnt signaling pathway plays fundamental roles in early development and in maintaining adult tissue homeostasis. R-spondin 3 (Rspo3) is a secreted protein that has been implicated in activating the Wnt/β-catenin signaling in amphibians and mammals. Here we report that zebrafish Rspo3 plays a negative role in regulating the zygotic Wnt/β-catenin signaling. Zebrafish Rspo3 has a unique domain structure. It contains a third furin-like (FU3) domain. This FU3 is present in other four ray-finned fish species studied but not in elephant shark. In zebrafish, rspo3 mRNA is maternally deposited and has a ubiquitous expression in early embryonic stages. After 12 hpf, its expression becomes tissue-specific. Forced expression of rspo3 promotes dorsoanterior patterning and increases the expression of dorsal and anterior marker genes. Knockdown of rspo3 increases ventral-posterior development and stimulates ventral and posterior marker genes expression. Forced expression of rspo3 abolishes exogenous Wnt3a action and reduces the endogenous Wnt signaling activity. Knockdown of rspo3 results in increased Wnt/β-catenin signaling activity. Further analyses indicate that Rspo3 does not promote maternal Wnt signaling. Human RSPO3 has similar action when tested in zebrafish embryos. These results suggest that Rspo3 regulates dorsoventral and anteroposterior patterning by negatively regulating the zygotic Wnt/β-catenin signaling in zebrafish embryos.

  2. 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

  3. Activation of the Syk tyrosine kinase is insufficient for downstream signal transduction in B lymphocytes

    PubMed Central

    Hsueh, Robert C; Hammill, Adrienne M; Lee, Jamie A; Uhr, Jonathan W; Scheuermann, Richard H

    2002-01-01

    Background Immature B lymphocytes and certain B cell lymphomas undergo apoptotic cell death following activation of the B cell antigen receptor (BCR) signal transduction pathway. Several biochemical changes occur in response to BCR engagement, including activation of the Syk tyrosine kinase. Although Syk activation appears to be necessary for some downstream biochemical and cellular responses, the signaling events that precede Syk activation remain ill defined. In addition, the requirements for complete activation of the Syk-dependent signaling step remain to be elucidated. Results A mutant form of Syk carrying a combination of a K395A substitution in the kinase domain and substitutions of three phenylalanines (3F) for the three C-terminal tyrosines was expressed in a murine B cell lymphoma cell line, BCL1.3B3 to interfere with normal Syk regulation as a means to examine the Syk activation step in BCR signaling. Introduction of this kinase-inactive mutant led to the constitutive activation of the endogenous wildtype Syk enzyme in the absence of receptor engagement through a 'dominant-positive' effect. Under these conditions, Syk kinase activation occurred in the absence of phosphorylation on Syk tyrosine residues. Although Syk appears to be required for BCR-induced apoptosis in several systems, no increase in spontaneous cell death was observed in these cells. Surprisingly, although the endogenous Syk kinase was enzymatically active, no enhancement in the phosphorylation of cytoplasmic proteins, including phospholipase Cγ2 (PLCγ2), a direct Syk target, was observed. Conclusion These data indicate that activation of Syk kinase enzymatic activity is insufficient for Syk-dependent signal transduction. This observation suggests that other events are required for efficient signaling. We speculate that localization of the active enzyme to a receptor complex specifically assembled for signal transduction may be the missing event. PMID:12470302

  4. The protist, Monosiga brevicollis, has a tyrosine kinase signaling network more elaborate and diverse than found in any known metazoan

    PubMed Central

    Manning, Gerard; Young, Susan L.; Miller, W. Todd; Zhai, Yufeng

    2008-01-01

    Tyrosine kinase signaling has long been considered a hallmark of intercellular communication, unique to multicellular animals. Our genomic analysis of the unicellular choanoflagellate Monosiga brevicollis discovers a remarkable count of 128 tyrosine kinases, 38 tyrosine phosphatases, and 123 phosphotyrosine (pTyr)-binding SH2 proteins, all higher counts than seen in any metazoan. This elaborate signaling network shows little orthology to metazoan counterparts yet displays many innovations reminiscent of metazoans. These include extracellular domains structurally related to those of metazoan receptor kinases, alternative methods for membrane anchoring and phosphotyrosine interaction in cytoplasmic kinases, and domain combinations that link kinases to small GTPase signaling and transcription. These proteins also display a wealth of combinations of known signaling domains. This uniquely divergent and elaborate signaling network illuminates the early evolution of pTyr signaling, explores innovative ways to traverse the cellular signaling circuitry, and shows extensive convergent evolution, highlighting pervasive constraints on pTyr signaling. PMID:18621719

  5. Antagonizing amyloid-β/calcium-sensing receptor signaling in human astrocytes and neurons: a key to halt Alzheimer's disease progression?

    PubMed Central

    Dal Prà, Ilaria; Chiarini, Anna; Armato, Ubaldo

    2015-01-01

    Astrocytes’ roles in late-onset Alzheimer's disease (LOAD) promotion are important, since they survive soluble or fibrillar amyloid-β peptides (Aβs) neurotoxic effects, undergo alterations of intracellular and intercellular Ca2+ signaling and gliotransmitters release via the Aβ/α7-nAChR (α7-nicotinic acetylcholine receptor) signaling, and overproduce/oversecrete newly synthesized Aβ42 oligomers, NO, and VEGF-A via the Aβ/CaSR (calcium-sensing receptor) signaling. Recently, it was suggested that the NMDAR (N-methyl-D-aspartate receptor) inhibitor nitromemantine would block the synapse-destroying effects of Aβ/α7-nAChR signaling. Yet, this and the progressive extracellular accrual and spreading of Aβ42 oligomers would be stopped well upstream by NPS 2143, an allosteric CaSR antagonist (calcilytic). PMID:25883618

  6. Disruption of cell-to-cell signaling does not abolish the antagonism of Phaeobacter gallaeciensis toward the fish pathogen Vibrio anguillarum in algal systems.

    PubMed

    Prol García, M J; D'Alvise, P W; Gram, L

    2013-09-01

    Quorum sensing (QS) regulates Phaeobacter gallaeciensis antagonism in broth systems; however, we demonstrate here that QS is not important for antagonism in algal cultures. QS mutants reduced Vibrio anguillarum to the same extent as the wild type. Consequently, a combination of probiotic Phaeobacter and QS inhibitors is a feasible strategy for aquaculture disease control.

  7. Herpes simplex virus-1 infection causes the secretion of a type I interferon-antagonizing protein and inhibits signaling at or before Jak-1 activation

    SciTech Connect

    Johnson, Karen E.; Knipe, David M.

    2010-01-05

    Host cells respond to viral infection by the production of type I interferons (IFNs), which induce the expression of antiviral genes. Herpes simplex virus I (HSV-1) encodes many mechanisms that inhibit the type I IFN response, including the ICP27-dependent inhibition of type I IFN signaling. Here we show inhibition of Stat-1 nuclear accumulation in cells that express ICP27. ICP27 expression also induces the secretion of a small, heat-stable type I IFN antagonizing protein that inhibits Stat-1 nuclear accumulation. We show that the inhibition of IFN-induced Stat-1 phosphorylation occurs at or upstream of Jak-1 phosphorylation. Finally, we show that ISG15 expression is induced after IFNalpha treatment in mock-infected cells, but not cells infected with WT HSV-1 or ICP27{sup -} HSV-1. These data suggest that HSV-1 has evolved multiple mechanisms to inhibit IFN signaling not only in infected cells, but also in neighboring cells, thereby allowing for increased viral replication and spread.

  8. 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

  9. Signaling, Regulation, and Specificity of the Type II p21-activated Kinases*

    PubMed Central

    Ha, Byung Hak; Morse, Elizabeth M.; Turk, Benjamin E.; Boggon, Titus J.

    2015-01-01

    The p21-activated kinases (PAKs) are a family of six serine/threonine kinases that act as key effectors of RHO family GTPases in mammalian cells. PAKs are subdivided into two groups: type I PAKs (PAK1, PAK2, and PAK3) and type II PAKs (PAK4, PAK5, and PAK6). Although these groups are involved in common signaling pathways, recent work indicates that the two groups have distinct modes of regulation and have both unique and common substrates. Here, we review recent insights into the molecular level details that govern regulation of type II PAK signaling. We also consider mechanisms by which signal transduction is regulated at the level of substrate specificity. Finally, we discuss the implications of these studies for clinical targeting of these kinases. PMID:25855792

  10. RKIP regulates MAP kinase signaling in cells with defective B-Raf activity.

    PubMed

    Zeng, Lingchun; Ehrenreiter, Karin; Menon, Jyotsana; Menard, Ray; Kern, Florian; Nakazawa, Yoko; Bevilacqua, Elena; Imamoto, Akira; Baccarini, Manuela; Rosner, Marsha Rich

    2013-05-01

    MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf kinase inhibitory protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism. Loss of B-Raf protein or activity significantly reduces MAPK activation in these cells. We show that RKIP depletion can rescue the compromised ERK activation and promote proliferation, and this rescue occurs through a Raf-1 dependent mechanism. These results provide formal evidence that RKIP is a bona fide regulator of Raf-1. We propose a new model in which RKIP plays a key role in regulating the ability of cells to signal through Raf-1 to ERK in B-Raf compromised cells.

  11. p70S6 kinase is a critical node that integrates HER-family and PI3 kinase signaling networks

    PubMed Central

    Axelrod, Mark J.; Gordon, Vicki; Mendez, Rolando E.; Leimgruber, Stephanie S.; Conaway, Mark R.; Sharlow, Elizabeth R.; Jameson, MarkJ.; Gioeli, Daniel G.; Weber, Michael J.

    2014-01-01

    Therapies targeting oncogenic drivers rapidly induce compensatory adaptive responses that blunt drug effectiveness, contributing to therapeutic resistance. Adaptive responses are characteristic of robust cell signaling networks, and thus there is increasing interest in drug combinations that co-target the driver and the adaptive response. An alternative approach to co-inhibiting oncogenic and adaptive targets is to identify a critical node where the activities of these targets converge. Nodes of convergence between signaling modules represent potential therapeutic vulnerabilities because their inhibition could result in collapse of the network, leading to enhanced cytotoxicity. In this report we demonstrate that p70S6 kinase (p70S6K) can function as a critical node linking HER-family and phosphoinositide-3-kinase (PI3K) pathway signaling. We used high-throughput combinatorial drug screening to identify adaptive survival responses to targeted therapies, and found that HER-family and PI3K represented compensatory signaling pathways. Co-targeting these pathways with drug combinations caused synergistic cytotoxicity in cases where inhibition of neither target was effective as a monotherapy. We utilized Reverse Phase Protein Arrays and determined that phosphorylation of ribosomal protein S6 was synergistically down-regulated upon HER-family and PI3K/mammalian target of rapamycin (mTOR) co-inhibition. Expression of constitutively active p70S6K protected against apoptosis induced by combined HER-family and PI3K/mTOR inhibition. Direct inhibition of p70S6K with small molecule inhibitors phenocopied HER-family and PI3K/mTOR co-inhibition. These data implicate p70S6K as a critical node in the HER-family/PI3K signaling network. The ability of direct inhibitors of p70S6K to phenocopy co-inhibition of two upstream signaling targets indicates that identification and targeting of critical nodes can overcome adaptive resistance to targeted therapies. PMID:24662264

  12. 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

  13. 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

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

    PubMed

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

    2015-11-26

    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.

  15. 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

  16. TLR signalling affects sperm mitochondrial function and motility via phosphatidylinositol 3-kinase and glycogen synthase kinase-3α.

    PubMed

    Zhu, Xingxing; Shi, Dongyan; Li, Xiaoqian; Gong, Weijuan; Wu, Fengjiao; Guo, Xuejiang; Xiao, Hui; Liu, Lixin; Zhou, Hong

    2016-03-01

    Infection in male and female genital tracts can lead to infertility. The underlying mechanisms of this process remain unclear. Toll-like receptors (TLRs) recognize conserved structures and respond to pathogens by initiating signals that activate inflammatory gene transcription. Here, we demonstrate that TLR activation in sperm reduces sperm motility via signalling through myeloid differentiation factor 88 (MyD88), phosphatidylinositol 3-kinase (PI3K), and glycogen synthase kinase (GSK)-3α. Upon TLR activation, phosphorylated forms of PI3K and GSK3α were detected in the mitochondria, and the mitochondrial membrane potential was impaired in sperm. In addition, mitochondrial ATP levels were decreased after TLR agonist stimulation. Furthermore, blocking PI3K or GSK3α activation abrogated these effects and reversed the TLR-induced reduction in sperm motility. These results identify a previously unrecognized TLR signalling pathway that leads to dysfunctional sperm mitochondria, which reduce sperm motility. Our study reveals a novel mechanism by which pathogenic infection affects sperm motility and possibly leads to infertility.

  17. 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...

  18. Antagonism between phytohormone signalling underlies the variation in disease susceptibility of tomato plants under elevated CO2

    PubMed Central

    Zhang, Shuai; Li, Xin; Sun, Zenghui; Shao, Shujun; Hu, Lingfei; Ye, Meng; Zhou, Yanhong; Xia, Xiaojian; Yu, Jingquan; Shi, Kai

    2015-01-01

    Increasing CO2 concentrations ([CO2]) have the potential to disrupt plant–pathogen interactions in natural and agricultural ecosystems, but the research in this area has often produced conflicting results. Variations in phytohormone salicylic acid (SA) and jasmonic acid (JA) signalling could be associated with variations in the responses of pathogens to plants grown under elevated [CO2]. In this study, interactions between tomato plants and three pathogens with different infection strategies were compared. Elevated [CO2] generally favoured SA biosynthesis and signalling but repressed the JA pathway. The exposure of plants to elevated [CO2] revealed a lower incidence and severity of disease caused by tobacco mosaic virus (TMV) and by Pseudomonas syringae, whereas plant susceptibility to necrotrophic Botrytis cinerea increased. The elevated [CO2]-induced and basal resistance to TMV and P. syringae were completely abolished in plants in which the SA signalling pathway nonexpressor of pathogenesis-related genes 1 (NPR1) had been silenced or in transgenic plants defective in SA biosynthesis. In contrast, under both ambient and elevated [CO2], the susceptibility to B. cinerea highly increased in plants in which the JA signalling pathway proteinase inhibitors (PI) gene had been silenced or in a mutant affected in JA biosynthesis. However, plants affected in SA signalling remained less susceptible to this disease. These findings highlight the modulated antagonistic relationship between SA and JA that contributes to the variation in disease susceptibility under elevated [CO2]. This information will be critical for investigating how elevated CO2 may affect plant defence and the dynamics between plants and pathogens in both agricultural and natural ecosystems. PMID:25657213

  19. MAP kinases with distinct inhibitory functions impart signaling specificity during yeast differentiation.

    PubMed

    Madhani, H D; Styles, C A; Fink, G R

    1997-11-28

    Filamentous invasive growth of S. cerevisiae requires multiple elements of the mitogen-activated protein kinase (MAPK) signaling cascade that are also components of the mating pheromone response pathway. Here we show that, despite sharing several constituents, the two pathways use different MAP kinases. The Fus3 MAPK regulates mating, whereas the Kss1 MAPK regulates filamentation and invasion. Remarkably, in addition to their kinase-dependent activation functions, Kss1 and Fus3 each have a distinct kinase-independent inhibitory function. Kss1 inhibits the filamentation pathway by interacting with its target transcription factor Ste12. Fus3 has a different inhibitory activity that prevents the inappropriate activation of invasion by the pheromone response pathway. In the absence of Fus3, there is erroneous crosstalk in which mating pheromone now activates filamentation-specific gene expression using the Kss1 MAPK. PMID:9393860

  20. 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

  1. Differences in Type I Interferon Signaling Antagonism by Dengue Viruses in Human and Non-Human Primate Cell Lines

    PubMed Central

    Medina, Freddy A.; Torres-Malavé, Giselle; Chase, Amanda J.; Santiago, Gilberto A.; Medina, Juan F.; Santiago, Luis M.; Muñoz-Jordán, Jorge L.

    2015-01-01

    Background/Objectives In vitro studies have shown that dengue virus (DENV) can thwart the actions of interferon (IFN)-α/β and prevent the development of an antiviral state in infected cells. Clinical studies looking at gene expression in patients with severe dengue show a reduced expression of interferon stimulated genes compared to patients with dengue fever. Interestingly, there are conflicting reports as to the ability of DENV or other flaviviruses to inhibit IFN-α/β signaling. Methodology/Principal Findings In order to determine the relative inhibition of IFN-α/β signaling by DENVs, a method combining flow cytometry and a four-parameter logistic regression model was established. A representative isolate from DENV-1, -3 and -4 and seventeen representative isolates encompassing all DENV-2 genotypes were evaluated. All of the DENVs evaluated in this study were capable of inhibiting IFN-α/β signaling. Most of the strains were able to inhibit IFN-α/β to a degree similar to DENV strain 16681; however, DENV-2 sylvatic strains demonstrated an increased inhibition of phosphorylated signal transducer and activator of transcription (pSTAT1). Surprisingly, we were unable to observe inhibition of pSTAT1 by DENV-2 sylvatic strains or the Asian strain 16681 in non-human primate (NHP) cell lines. Analysis in primary Rhesus macaque dendritic cells suggests that DENVs are capable of inhibiting IFN signaling in these cells. However, contrary to human dendritic cells, production of IFN-α was detected in the supernatant of DENV-infected Rhesus macaque dendritic cells. Conclusions The ability of DENVs to inhibit IFN-α/β signaling is conserved. Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes. DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific phenomena or due to

  2. A peptide targeting an interaction interface disrupts the dopamine D1-D2 receptor heteromer to block signaling and function in vitro and in vivo: effective selective antagonism

    PubMed Central

    Hasbi, Ahmed; Perreault, Melissa L.; Shen, Maurice Y. F.; Zhang, Lucia; To, Ryan; Fan, Theresa; Nguyen, Tuan; Ji, Xiaodong; O'Dowd, Brian F.; George, Susan R.

    2014-01-01

    Although the dopamine D1-D2 receptor heteromer has emerging physiological relevance and a postulated role in different neuropsychiatric disorders, such as drug addiction, depression, and schizophrenia, there is a need for pharmacological tools that selectively target such receptor complexes in order to analyze their biological and pathophysiological functions. Since no selective antagonists for the D1-D2 heteromer are available, serial deletions and point mutations were used to precisely identify the amino acids involved in an interaction interface between the receptors, residing within the carboxyl tail of the D1 receptor that interacted with the D2 receptor to form the D1-D2 receptor heteromer. It was determined that D1 receptor carboxyl tail residues 404Glu and 405Glu were critical in mediating the interaction with the D2 receptor. Isolated mutation of these residues in the D1 receptor resulted in the loss of agonist activation of the calcium signaling pathway mediated through the D1-D2 receptor heteromer. The physical interaction between the D1 and D2 receptor could be disrupted, as shown by coimmunoprecipitation and BRET analysis, by a small peptide generated from the D1 receptor sequence that contained these amino acids, leading to a switch in G-protein affinities and loss of calcium signaling, resulting in the inhibition of D1-D2 heteromer function. The use of the D1-D2 heteromer-disrupting peptide in vivo revealed a pathophysiological role for the D1-D2 heteromer in the modulation of behavioral despair. This peptide may represent a novel pharmacological tool with potential therapeutic benefits in depression treatment.—Hasbi, A., Perreault, M. L., Shen, M. Y. F., Zhang, L., To, R., Fan, T., Nguyen, T., Ji, X., O'Dowd, B. F., George, S. R. A peptide targeting an interaction interface disrupts the dopamine D1-D2 receptor heteromer to block signaling and function in vitro and in vivo: effective selective antagonism. PMID:25063849

  3. Histone deacetylase HDA6 enhances brassinosteroid signaling by inhibiting the BIN2 kinase.

    PubMed

    Hao, Yuhan; Wang, Haijiao; Qiao, Shenglong; Leng, Linna; Wang, Xuelu

    2016-09-13

    Glycogen synthase kinase 3 (GSK3)-like kinases play important roles in brassinosteroid (BR), abscisic acid, and auxin signaling to regulate many aspects of plant development and stress responses. The Arabidopsis thaliana GSK3-like kinase BR-INSENSITIVE 2 (BIN2) acts as a key negative regulator in the BR signaling pathway, but the mechanisms regulating BIN2 function remain unclear. Here we report that the histone deacetylase HDA6 can interact with and deacetylate BIN2 to repress its kinase activity. The hda6 mutant showed a BR-repressed phenotype in the dark and was less sensitive to BR biosynthesis inhibitors. Genetic analysis indicated that HDA6 regulates BR signaling through BIN2. Furthermore, we identified K189 of BIN2 as an acetylated site, which can be deacetylated by HDA6 to influence BIN2 activity. Glucose can affect the acetylation level of BIN2 in plants, indicating a connection to cellular energy status. These findings provide significant insights into the regulation of GSK3-like kinases in plant growth and development. PMID:27562168

  4. Antagonism between MyD88- and TRIF-dependent signals in B7RP-1 up-regulation.

    PubMed

    Zhou, Zuping; Hoebe, Kasper; Du, Xin; Jiang, Zhengfan; Shamel, Louis; Beutler, Bruce

    2005-06-01

    Type I interferons (IFN) play a critical role in the Toll-like receptor (TLR)-mediated expression of B7 costimulatory family members. For example, LPS-induced up-regulation of CD80 (B7.1) and CD86 (B7.2) is abrogated in antigen-presenting cells (APC) deficient in TRIF or TRAM, two adaptors that are responsible for TLR4-mediated production of Type I IFN. In this report, we demonstrate that LPS-induced up-regulation of B7-related protein 1 (B7RP-1), a ligand for ICOS, is dependent primarily upon the MyD88-dependent signaling pathway. Signaling via the TRIF pathway sharply limits MyD88-dependent B7RP-1 up-regulation. Hence, LPS induces significantly higher B7RP-1 expression on TRIF- or TRAM-deficient mouse peritoneal macrophages and on TRIF-deficient mouse splenic B cells as compared to wild-type cells. Further studies reveal that Type I IFN are general suppressors of TLR-mediated up-regulation of B7RP-1. These data indicate that Type I IFN play a dual role in the TLR-mediated expression of B7 costimulatory family members and suggest that they may act to limit B7RP-1 expression and thus limit signals derived from B7RP-1-ICOS interaction.

  5. c-Yes tyrosine kinase is a potent suppressor of ES cell differentiation and antagonizes the actions of its closest phylogenetic relative, c-Src.

    PubMed

    Zhang, Xiong; Meyn, Malcolm A; Smithgall, Thomas E

    2014-01-17

    Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst stage embryo and are characterized by self-renewal and pluripotency. Previous work has shown that Src-family tyrosine kinases display dynamic expression and activity changes during ES cell differentiation, suggesting distinct functions in the control of developmental fate. Here we used ES cells to test the hypothesis that c-Src and its closest phylogenetic relative, c-Yes, act in biological opposition despite their strong homology. Unlike c-Src, enforced expression of active c-Yes blocked ES cell differentiation to embryoid bodies by maintaining pluripotency gene expression. To explore the interplay of c-Src and c-Yes in ES cell differentiation, we engineered c-Src and c-Yes mutants that are resistant to A-419259, a potent pyrrolopyrimidine inhibitor of the Src kinase family. Previous studies have shown that A-419259 treatment blocks all Src-family kinase activity in ES cells, preventing differentiation while maintaining pluripotency. Expression of inhibitor-resistant c-Src but not c-Yes rescued the A-419259 differentiation block, resulting in a cell population with properties of both primitive ectoderm and endoderm. Remarkably, when inhibitor-resistant c-Src and c-Yes were expressed together in ES cells, c-Yes activity suppressed c-Src-mediated differentiation. These studies show that even closely related kinases such as c-Src and c-Yes have unique and opposing functions in the same cell type. Selective agonists or inhibitors of c-Src versus c-Yes activity may allow more precise pharmacological manipulation of ES cell fate and have broader applications in other biological systems that express multiple Src family members such as tumor cells.

  6. Smoothened Signaling in Vertebrates Is Facilitated by a G Protein-coupled Receptor Kinase

    PubMed Central

    Philipp, Melanie; Fralish, Gregory B.; Meloni, Alison R.; Chen, Wei; MacInnes, Alyson W.; Barak, Lawrence S.

    2008-01-01

    Smoothened, a heptahelical membrane protein, functions as the transducer of Hedgehog signaling. The kinases that modulate Smoothened have been thoroughly analyzed in flies. However, little is known about how phosphorylation affects Smoothened in vertebrates, mainly, because the residues, where Smoothened is phosphorylated are not conserved from Drosophila to vertebrates. Given its molecular architecture, Smoothened signaling is likely to be regulated in a manner analogous to G protein–coupled receptors (GPCRs). Previously, it has been shown, that arrestins and GPCR kinases, (GRKs) not only desensitize G protein–dependent receptor signaling but also function as triggers for GPCR trafficking and formation of signaling complexes. Here we describe that a GRK contributes to Smoothened-mediated signaling in vertebrates. Knockdown of the zebrafish homolog of mammalian GRK2/3 results in lowered Hedgehog transcriptional responses, impaired muscle development, and neural patterning. Results obtained in zebrafish are corroborated both in cell culture, where zGRK2/3 phosphorylates Smoothened and promotes Smoothened signal transduction and in mice where deletion of GRK2 interferes with neural tube patterning. Together, these data suggest that a GRK functions as a vertebrate kinase for Smoothened, promoting Hedgehog signal transduction during early development. PMID:18815277

  7. Crystal structures of the phosphorylated BRI1 kinase domain and implications for brassinosteroid signal initiation

    PubMed Central

    Bojar, Daniel; Martinez, Jacobo; Santiago, Julia; Rybin, Vladimir; Bayliss, Richard; Hothorn, Michael

    2014-01-01

    Brassinosteroids, which control plant growth and development, are sensed by the membrane receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1). Brassinosteroid binding to the BRI1 leucine-rich repeat (LRR) domain induces heteromerisation with a SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK)-family co-receptor. This process allows the cytoplasmic kinase domains of BRI1 and SERK to interact, trans-phosphorylate and activate each other. Here we report crystal structures of the BRI1 kinase domain in its activated form and in complex with nucleotides. BRI1 has structural features reminiscent of both serine/threonine and tyrosine kinases, providing insight into the evolution of dual-specificity kinases in plants. Phosphorylation of Thr1039, Ser1042 and Ser1044 causes formation of a catalytically competent activation loop. Mapping previously identified serine/threonine and tyrosine phosphorylation sites onto the structure, we analyse their contribution to brassinosteroid signaling. The location of known genetic missense alleles provide detailed insight into the BRI1 kinase mechanism, while our analyses are inconsistent with a previously reported guanylate cyclase activity. We identify a protein interaction surface on the C-terminal lobe of the kinase and demonstrate that the isolated BRI1, SERK2 and SERK3 cytoplasmic segments form homodimers in solution and have a weak tendency to heteromerise. We propose a model in which heterodimerisation of the BRI1 and SERK ectodomains brings their cytoplasmic kinase domains in a catalytically competent arrangement, an interaction that can be modulated by the BRI1 inhibitor protein BKI1. PMID:24461462

  8. A membrane-anchored protein kinase involved in Brassica self-incompatibility signaling.

    PubMed

    Murase, Kohji; Shiba, Hiroshi; Iwano, Megumi; Che, Fang-Sik; Watanabe, Masao; Isogai, Akira; Takayama, Seiji

    2004-03-01

    Self-incompatibility (SI) response in Brassica is initiated by haplotype-specific interactions between the pollen-borne ligand S locus protein 11/SCR and its stigmatic S receptor kinase, SRK. This binding induces autophosphorylation of SRK, which is then thought to trigger a signaling cascade that leads to self-pollen rejection. A recessive mutation of the modifier (m) gene eliminates the SI response in stigma. Positional cloning of M has revealed that it encodes a membrane-anchored cytoplasmic serine/threonine protein kinase, designated M locus protein kinase (MLPK). Transient expression of MLPK restores the ability of mm papilla cells to reject self-pollen, suggesting that MLPK is a positive mediator of Brassica SI signaling.

  9. 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

  10. eyelid antagonizes wingless signaling during Drosophila development and has homology to the Bright family of DNA-binding proteins

    PubMed Central

    Treisman, Jessica E.; Luk, Alvin; Rubin, Gerald M.; Heberlein, Ulrike

    1997-01-01

    In Drosophila, pattern formation at multiple stages of embryonic and imaginal development depends on the same intercellular signaling pathways. We have identified a novel gene, eyelid (eld), which is required for embryonic segmentation, development of the notum and wing margin, and photoreceptor differentiation. In these tissues, eld mutations have effects opposite to those caused by wingless (wg) mutations. eld encodes a widely expressed nuclear protein with a region homologous to a novel family of DNA-binding domains. Based on this homology and on the phenotypic analysis, we suggest that Eld could act as a transcription factor antagonistic to the Wg pathway. PMID:9271118

  11. 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

  12. 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.

  13. Regulation of Yeast G Protein Signaling by the Kinases That Activate the AMPK Homolog Snf1

    PubMed Central

    Clement, Sarah T.; Dixit, Gauri; Dohlman, Henrik G.

    2014-01-01

    Extracellular signals, such as nutrients and hormones, cue intracellular pathways to produce adaptive responses. Often, cells must coordinate their responses to multiple signals to produce an appropriate outcome. We showed that components of a glucose-sensing pathway acted on components of a heterotrimeric guanine nucleotide–binding protein (G protein)–mediated pheromone signaling pathway in the yeast Saccharomyces cerevisiae. We demonstrated that the G protein α subunit Gpa1 was phosphorylated in response to conditions of reduced glucose availability and that this phosphorylation event contributed to reduced pheromone-dependent stimulation of mitogen-activated protein kinases, gene transcription, cell morphogenesis, and mating efficiency. We found that Elm1, Sak1, and Tos3, the kinases that phosphorylate Snf1, the yeast homolog of adenosine monophosphate–activated protein kinase (AMPK), in response to limited glucose availability, also phosphorylated Gpa1 and contributed to the diminished mating response. Reg1, the regulatory subunit of the phosphatase PP1 that acts on Snf1, was likewise required to reverse the phosphorylation of Gpa1 and to maintain the mating response. Thus, the same kinases and phosphatase that regulate Snf1 also regulate Gpa1. More broadly, these results indicate that the pheromone signaling and glucose-sensing pathways communicate directly to coordinate cell behavior. PMID:24003255

  14. 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.

  15. CD28 signal transduction pathways. A comparison of B7-1 and B7-2 regulation of the map kinases: ERK2 and Jun kinases.

    PubMed

    Nunes, J A; Battifora, M; Woodgett, J R; Truneh, A; Olive, D; Cantrell, D A

    1996-01-01

    The present study compares the mitogen-activated protein (MAP) kinase responses in T cells activated with the CD28 ligands B7-1 (CD80) and B7-2/B70 (CD86). Ligands B7-1 and B7-2 do not activate the Raf-1/ERK2 cascade, but share the ability to activate related Jun kinases. These natural ligands for CD28 had no stimulatory effect alone on Jun kinase activation, but the data show that B7-1 and B7-2 could both co-operate with intracellular Ca2+ increase and protein kinase C (PKC) activation to stimulate Jun kinases. The present study shows that the interaction of CD28 with its ligands B7-1 and B7-2 can induce identical signal transduction through the MAP kinase cascades.

  16. p21ras initiates Rac-1 but not phosphatidyl inositol 3 kinase/PKB, mediated signaling pathways in T lymphocytes.

    PubMed

    Genot, E; Reif, K; Beach, S; Kramer, I; Cantrell, D

    1998-10-01

    p21ras is activated by the T cell antigen receptor (TCR) and then co-ordinates important signaling pathways for T lymphocyte activation. Effector pathways for this guanine nucleotide binding protein in T cells are mediated by the serine/threonine kinase Raf-1 and the Ras-related GTPase Rac-1. In fibroblasts, an important effector for the Ras oncogene is Phosphatidylinositol 3-kinase (PtdIns 3-kinase). Activation of this lipid kinase is able to induce critical Rac-1 signaling pathways and can couple p21ras to cell survival mechanisms via the serine/threonine kinase Akt/PKB. The role of PtdIns 3-kinase in Ras signaling in T cells has not been explored. In the present study, we examined the ability of PtdIns 3-kinase to initiate the Rac-1 signaling pathways important for T cell activation. We also examined the possibility that Akt/PKB is regulated by Ras signaling pathways in T lymphocytes. The results show that Ras can initiate a Rac-1 mediated pathway that regulates the transcriptional function of AP-1 complexes. PtdIns 3-kinase signals cannot mimic p21ras and induce the Rac mediated responses of AP-1 transcriptional activation. Moreover, neither TCR or Ras activation of AP-1 is dependent on PtdIns 3-kinase. PKB is activated in response to triggering of the T cell antigen receptor; PtdIns 3-kinase activity is both required and sufficient for this TCR response. In contrast, p21ras signals are unable to induce Akt/PKB activity in T cell nor is Ras function required for Akt/PKB activation in response to the TCR. The present data thus highlight that PtdIns 3-kinase and Akt/PKB are not universal Ras effector molecules. Ras can initiate Rac-1 regulated signaling pathways in the context of T cell antigen receptor function independently of PtdIns 3-kinase activity.

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

    PubMed

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

    2015-03-01

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

  18. 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

  19. Regulation of mRNA export by the PI3 kinase/AKT signal transduction pathway

    PubMed Central

    Quaresma, Alexandre Jose Christino; Sievert, Rachel; Nickerson, Jeffrey A.

    2013-01-01

    UAP56, ALY/REF, and NXF1 are mRNA export factors that sequentially bind at the 5′ end of a nuclear mRNA but are also reported to associate with the exon junction complex (EJC). To screen for signal transduction pathways regulating mRNA export complex assembly, we used fluorescence recovery after photobleaching to measure the binding of mRNA export and EJC core proteins in nuclear complexes. The fraction of UAP56, ALY/REF, and NXF1 tightly bound in complexes was reduced by drug inhibition of the phosphatidylinositide 3-kinase (PI3 kinase)/AKT pathway, as was the tightly bound fraction of the core EJC proteins eIF4A3, MAGOH, and Y14. Inhibition of the mTOR mTORC1 pathway decreased the tight binding of MAGOH. Inhibition of the PI3 kinase/AKT pathway increased the export of poly(A) RNA and of a subset of candidate mRNAs. A similar effect of PI3 kinase/AKT inhibition was observed for mRNAs from both intron-containing and intronless histone genes. However, the nuclear export of mRNAs coding for proteins targeted to the endoplasmic reticulum or to mitochondria was not affected by the PI3 kinase/AKT pathway. These results show that the active PI3 kinase/AKT pathway can regulate mRNA export and promote the nuclear retention of some mRNAs. PMID:23427269

  20. 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

  1. Extracellular Signal-Regulated Kinase Activates Topoisomerase IIα through a Mechanism Independent of Phosphorylation

    PubMed Central

    Shapiro, Paul S.; Whalen, Anne M.; Tolwinski, Nicholas S.; Wilsbacher, Julie; Froelich-Ammon, Stacie J.; Garcia, Marileila; Osheroff, Neil; Ahn, Natalie G.

    1999-01-01

    The mitogen-activated protein (MAP) kinases, extracellular signal-related kinase 1 (ERK1) and ERK2, regulate cellular responses by mediating extracellular growth signals toward cytoplasmic and nuclear targets. A potential target for ERK is topoisomerase IIα, which becomes highly phosphorylated during mitosis and is required for several aspects of nucleic acid metabolism, including chromosome condensation and daughter chromosome separation. In this study, we demonstrated interactions between ERK2 and topoisomerase IIα proteins by coimmunoprecipitation from mixtures of purified enzymes and from nuclear extracts. In vitro, diphosphorylated active ERK2 phosphorylated topoisomerase IIα and enhanced its specific activity by sevenfold, as measured by DNA relaxation assays, whereas unphosphorylated ERK2 had no effect. However, activation of topoisomerase II was also observed with diphosphorylated inactive mutant ERK2, suggesting a mechanism of activation that depends on the phosphorylation state of ERK2 but not on its kinase activity. Nevertheless, activation of ERK by transient transfection of constitutively active mutant MAP kinase kinase 1 (MKK1) enhanced endogenous topoisomerase II activity by fourfold. Our findings indicate that ERK regulates topoisomerase IIα in vitro and in vivo, suggesting a potential target for the MKK/ERK pathway in the modulation of chromatin reorganization events during mitosis and in other phases of the cell cycle. PMID:10207078

  2. Anti-apoptotic signaling of pleiotrophin through its receptor, anaplastic lymphoma kinase.

    PubMed

    Bowden, Emma T; Stoica, Gerald E; Wellstein, Anton

    2002-09-27

    The secreted growth factor pleiotrophin (PTN) can induce mitogenesis in cells that express the receptor for this growth factor, anaplastic lymphoma kinase (ALK). Here we examine the ability of PTN to produce anti-apoptotic signals. We demonstrate that PTN is a survival factor for SW-13 epithelial cells and show that ribozyme-mediated depletion of ALK from SW-13 cells abolishes this effect of PTN. Furthermore, in serum-starved NIH3T3 fibroblasts PTN prevents apoptosis (measured by annexin V staining) with an EC(50) of 0.2 ng/ml and induces cell growth at higher concentrations of PTN. A polyclonal antibody against the PTN ligand-binding domain of the ALK receptor (alpha-LBD) was a partial agonist for ALK in NIH3T3 cells. This alpha-LBD antibody showed high agonist activity for anti-apoptosis (56 +/- 9% relative to PTN), low agonist activity for cell growth (21 +/- 1% relative to PTN), and was an antagonist of PTN-induced cell growth (61 +/- 2% inhibition). Both MAP kinase and phosphatidylinositol (PI) 3-kinase cascades in NIH3T3 cells were activated by PTN, and this effect persisted for up to 3 h. Surprisingly, the anti-apoptotic effect of PTN was completely blocked by the MAP kinase inhibitor UO126, but was not affected by the PI 3-kinase inhibitor LY294002. In contrast, PTN-dependent cell growth required both MAPK and PI 3-kinase activity. We conclude that anti-apoptotic signaling of PTN through ALK in NIH3T3 fibroblasts is via the MAP kinase pathway.

  3. 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.

  4. 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

  5. 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

  6. 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

  7. Anti-Sclerostin antibody inhibits internalization of Sclerostin and Sclerostin-mediated antagonism of Wnt/LRP6 signaling.

    PubMed

    van Dinther, Maarten; Zhang, Juan; Weidauer, Stella E; Boschert, Verena; Muth, Eva-Maria; Knappik, Achim; de Gorter, David J J; van Kasteren, Puck B; Frisch, Christian; Mueller, Thomas D; ten Dijke, Peter

    2013-01-01

    Sclerosteosis is a rare high bone mass disease that is caused by inactivating mutations in the SOST gene. Its gene product, Sclerostin, is a key negative regulator of bone formation and might therefore serve as a target for the anabolic treatment of osteoporosis. The exact molecular mechanism by which Sclerostin exerts its antagonistic effects on Wnt signaling in bone forming osteoblasts remains unclear. Here we show that Wnt3a-induced transcriptional responses and induction of alkaline phosphatase activity, an early marker of osteoblast differentiation, require the Wnt co-receptors LRP5 and LRP6. Unlike Dickkopf1 (DKK1), Sclerostin does not inhibit Wnt-3a-induced phosphorylation of LRP5 at serine 1503 or LRP6 at serine 1490. Affinity labeling of cell surface proteins with [(125)I]Sclerostin identified LRP6 as the main specific Sclerostin receptor in multiple mesenchymal cell lines. When cells were challenged with Sclerostin fused to recombinant green fluorescent protein (GFP) this was internalized, likely via a Clathrin-dependent process, and subsequently degraded in a temperature and proteasome-dependent manner. Ectopic expression of LRP6 greatly enhanced binding and cellular uptake of Sclerostin-GFP, which was reduced by the addition of an excess of non-GFP-fused Sclerostin. Finally, an anti-Sclerostin antibody inhibited the internalization of Sclerostin-GFP and binding of Sclerostin to LRP6. Moreover, this antibody attenuated the antagonistic activity of Sclerostin on canonical Wnt-induced responses.

  8. Uric acid induces oxidative stress and growth inhibition by activating adenosine monophosphate-activated protein kinase and extracellular signal-regulated kinase signal pathways in pancreatic β cells.

    PubMed

    Zhang, Yongneng; Yamamoto, Tetsuya; Hisatome, Ichiro; Li, Youfeng; Cheng, Weijie; Sun, Ning; Cai, Bozhi; Huang, Tianliang; Zhu, Yuzhang; Li, Zhi; Jing, Xubin; Zhou, Rui; Cheng, Jidong

    2013-08-15

    Hyperuricaemia is a disorder of purine metabolism, and is strongly associated with insulin resistance and abnormal glucose metabolism. As the producer of insulin, pancreatic β cells might be affected by elevated serum uric acid levels and contribute to the disregulated glucose metabolism. In this study, we investigated the effect of high uric acid on rat pancreatic β cell function. Under high uric acid condition, proliferation of pancreatic β cells was inhibited, production of reactive oxygen species increased, and glucose stimulated insulin secretion was also compromised. Further examination on signal transduction pathways revealed that uric acid-induced ROS is involved in the activation of adenosine monophosphate-activated protein kinase (AMPK), and extracellular signal-regulated kinase (ERK). Pharmacological inhibition of ERK activation rescued β cells from growth inhibition. More importantly, activation of ERK induced by uric acid is significantly diminished by AMPK inhibitor, indicating ERK as a downstream target of AMPK in response to high uric acid condition. We also investigated the transportation channel for uric acid into pancreatic β cells. While major urate transporter URAT1 is not expressed in β cells, organic anion transporter (OAT) inhibitor successfully blocked the activation of ERK by uric acid. Our data indicate that high uric acid levels induce oxidative damage and inhibit growth of rat pancreatic β cells by activating the AMPK and ERK signal pathways. Hyperuricemia may contribute to abnormal glucose metabolism by causing oxidative damage and function inhibition of pancreatic β cells.

  9. The forced swimming-induced behavioural immobility response involves histone H3 phospho-acetylation and c-Fos induction in dentate gyrus granule neurons via activation of the N-methyl-D-aspartate/extracellular signal-regulated kinase/mitogen- and stress-activated kinase signalling pathway.

    PubMed

    Chandramohan, Yalini; Droste, Susanne K; Arthur, J Simon C; Reul, Johannes M H M

    2008-05-01

    The hippocampus is involved in learning and memory. Previously, we have shown that the acquisition of the behavioural immobility response after a forced swim experience is associated with chromatin modifications and transcriptional induction in dentate gyrus granule neurons. Given that both N-methyl-D-aspartate (NMDA) receptors and the extracellular signal-regulated kinases (ERK) 1/2 signalling pathway are involved in neuroplasticity processes underlying learning and memory, we investigated in rats and mice whether these signalling pathways regulate chromatin modifications and transcriptional events participating in the acquisition of the immobility response. We found that: (i) forced swimming evoked a transient increase in the number of phospho-acetylated histone H3-positive [P(Ser10)-Ac(Lys14)-H3(+)] neurons specifically in the middle and superficial aspects of the dentate gyrus granule cell layer; (ii) antagonism of NMDA receptors and inhibition of ERK1/2 signalling blocked forced swimming-induced histone H3 phospho-acetylation and the acquisition of the behavioural immobility response; (iii) double knockout (DKO) of the histone H3 kinase mitogen- and stress-activated kinases (MSK) 1/2 in mice completely abolished the forced swimming-induced increases in histone H3 phospho-acetylation and c-Fos induction in dentate granule neurons and the behavioural immobility response; (iv) blocking mineralocorticoid receptors, known not to be involved in behavioural immobility in the forced swim test, did not affect forced swimming-evoked histone H3 phospho-acetylation in dentate neurons; and (v) the pharmacological manipulations and gene deletions did not affect behaviour in the initial forced swim test. We conclude that the forced swimming-induced behavioural immobility response requires histone H3 phospho-acetylation and c-Fos induction in distinct dentate granule neurons through recruitment of the NMDA/ERK/MSK 1/2 pathway.

  10. Lovastatin inhibits the extracellular-signal-regulated kinase pathway in immortalized rat brain neuroblasts

    PubMed Central

    Cerezo-Guisado, Maria Isabel; GarcíA-Román, Natalia; García-MaríN, Luis Jesús; Álvarez-Barrientos, Alberto; Bragado, Maria Julia; Lorenzo, Maria Jesús

    2006-01-01

    We have shown previously that lovastatin, a 3-hydroxy-3-methyl- glutaryl coenzyme A reductase inhibitor, induces apoptosis in spontaneously immortalized rat brain neuroblasts. In the present study, we analysed the intracellular signal transduction pathways by which lovastatin induces neuroblast apoptosis. We showed that lovastatin efficiently inhibited Ras activation, which was associ-ated with a significant decrease in ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Lovastatin also decreased CREB phosphorylation and CREB-mediated gene expression. The effects of lovastatin on the Ras/ERK1/2/CREB pathway were time- and concentration-dependent and fully prevented by meva-lonate. In addition, we showed that two MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] inhibitors, PD98059 and PD184352, were poor inducers of apoptosis in serum-treated neuroblasts. However, these inhibitors significantly increased apop-tosis induced by lovastatin treatment. Furthermore, we showed that pharmacological inhibition of both MEK and phosphoinos-itide 3-kinase activities was able to induce neuroblast apoptosis with similar efficacy as lovastatin. Our results suggest that lovast-atin triggers neuroblast apoptosis by regulating several signalling pathways, including the Ras/ERK1/2 pathway. These findings might also contribute to elucidate the intracellular mechanisms involved in the central nervous system side effects associated with statin therapy. PMID:16952276

  11. Mitochondrial integrity: preservation through Akt/Pim-1 kinase signaling in the cardiomyocyte

    PubMed Central

    Sussman, Mark A

    2014-01-01

    The central role of mitochondria as mediators of cell survival is indisputable and gathering increasing attention as a focal point for interventional strategies to mitigate apoptotic cell death in the wake of cardiomyopathic injury. A legacy of signal transduction studies has proven that mitochondrial integrity can be enhanced by kinases involved in cell survival. Among the many survival signaling cascades under investigation, the wide-ranging impact of Akt upon mitochondrial biology is well known. However, despite years of investigation, emerging research continues to reveal new mechanisms governing the protective effects of Akt signaling in the context of cardiomyocyte mitochondria. This review focuses on two emerging pathways that mediate preservation of mitochondrial function downstream of Akt: hexokinase and Pim-1 kinase. PMID:19673671

  12. Specificity of MAP kinase signaling in yeast differentiation involves transient versus sustained MAPK activation.

    PubMed

    Sabbagh, W; Flatauer, L J; Bardwell, A J; Bardwell, L

    2001-09-01

    Signals transmitted by common components often elicit distinct (yet appropriate) outcomes. In yeast, two developmental options-mating and invasive growth-are both regulated by the same MAP kinase cascade. Specificity has been thought to result from specialized roles for the two MAP kinases, Kss1 and Fus3, and because Fus3 prevents Kss1 from gaining access to the mating pathway. Kss1 has been thought to participate in mating only when Fus3 is absent. Instead, we show that Kss1 is rapidly phosphorylated and potently activated by mating pheromone in wild-type cells, and that this is required for normal pheromone-induced gene expression. Signal identity is apparently maintained because active Fus3 limits the extent of Kss1 activation, thereby preventing inappropriate signal crossover. PMID:11583629

  13. Ethanol activates midkine and anaplastic lymphoma kinase signaling in neuroblastoma cells and in the brain.

    PubMed

    He, Donghong; Chen, Hu; Muramatsu, Hisako; Lasek, Amy W

    2015-11-01

    Alcohol engages signaling pathways in the brain. Midkine (MDK) is a neurotrophic factor that is over-expressed in the prefrontal cortex of alcoholics. MDK and one of its receptors, anaplastic lymphoma kinase (ALK), also regulate behavioral responses to ethanol in mice. The goal of this study was to determine whether MDK and ALK expression and signaling are activated by ethanol. We found that ethanol treatment of neuroblastoma cells increased MDK and ALK expression. We also assessed activation of ALK by ethanol in cells and found that ALK and ALK-dependent extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3) phosphorylation increased rapidly with ethanol exposure. Similarly, treatment of cells with recombinant MDK protein increased ALK, ERK and STAT3 phosphorylation, suggesting that ethanol may utilize MDK to activate ALK signaling. In support of this, transfection of cells with MDK siRNAs attenuated ALK signaling in response to ethanol. Ethanol also activates ERK signaling in the brain. We found that inhibition of ALK or knockout of MDK attenuated ethanol-induced ERK phosphorylation in mouse amygdala. These results demonstrate that ethanol engages MDK and ALK signaling, which has important consequences for alcohol-induced neurotoxicity and the regulation of behaviors related to alcohol abuse.

  14. ACK1/TNK2 tyrosine kinase: molecular signaling and evolving role in cancers.

    PubMed

    Mahajan, K; Mahajan, N P

    2015-08-01

    Deregulated tyrosine kinase signaling alters cellular homeostasis to drive cancer progression. The emergence of a non-receptor tyrosine kinase (non-RTK), ACK1 (also known as activated Cdc42-associated kinase 1 or TNK2) as an oncogenic kinase, has uncovered novel mechanisms by which tyrosine kinase signaling promotes cancer progression. Although early studies focused on ACK1 as a cytosolic effector of activated transmembrane 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, which modifies KDM3A by tyrosine phosphorylation to regulate the 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.

  15. 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

  16. Specificity is complex and time consuming: mutual exclusivity in tyrosine kinase-mediated signaling.

    PubMed

    O'Rourke, Lisa; Ladbury, John E

    2003-06-01

    Most fundamental cellular processes are transduced through tyrosine kinase (TK)-mediated pathways. For transduction without corruption, the protein-protein interactions involved have to be mutually exclusive. Many of these proteins bind via homologous domains whose binding characteristics suggest that their innate specificity is not sufficiently high to account for the integrity of signal transduction. Stimulation of TK-mediated signals is often accompanied by recruitment of a precise, multimolecular protein complex that is itself capable of imposing specificity. Furthermore, this complex provides protection against phosphatase activity, controlling the longevity of the active signaling complex, and thus influencing outcomes in subsequent downstream events.

  17. Regulation and function of syk tyrosine kinase in mast cell signaling and beyond.

    PubMed

    de Castro, Rodrigo Orlandini

    2011-01-01

    The protein tyrosine kinase Syk plays a critical role in FcεRI signaling in mast cells. Binding of Syk to phosphorylated immunoreceptor tyrosine-based activation motifs (p-ITAM) of the receptor subunits results in conformational changes and tyrosine phosphorylation at multiple sites that leads to activation of Syk. The phosphorylated tyrosines throughout the molecule play an important role in the regulation of Syk-mediated signaling. Reconstitution of receptor-mediated signaling in Syk(-/-) cells by wild-type Syk or mutants which have substitution of these tyrosines with phenylalanine together with in vitro assays has been useful strategies to understand the regulation and function of Syk.

  18. 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.

  19. 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.

  20. The Potential for Signal Integration and Processing in Interacting Map Kinase Cascades

    PubMed Central

    Schwacke, John H.; Voit, Eberhard O.

    2009-01-01

    The cellular response to environmental stimuli requires biochemical information processing through which sensory inputs and cellular status are integrated and translated into appropriate responses by way of interacting networks of enzymes. One such network, the Mitogen Activated Protein (MAP) kinase cascade is a highly conserved signal transduction module that propagates signals from cell surface receptors to various cytosolic and nuclear targets by way of a phosphorylation cascade. We have investigated the potential for signal processing within a network of interacting feed-forward kinase cascades typified by the MAP kinase cascade. A genetic algorithm was used to search for sets of kinetic parameters demonstrating representative key input-output patterns of interest. We discuss two of the networks identified in our study, one implementing the exclusive-or function (XOR) and another implementing what we refer to as an in-band detector (IBD) or two-sided threshold. These examples confirm the potential for logic and amplitude-dependent signal processing in interacting MAP kinase cascades demonstrating limited cross-talk. Specifically, the XOR function allows the network to respond to either one, but not both signals simultaneously, while the IBD permits the network to respond exclusively to signals within a given range of strength, and to suppress signals below as well as above this range. The solution to the XOR problem is interesting in that it requires only two interacting pathways, crosstalk at only one layer, and no feedback or explicit inhibition. These types of responses are not only biologically relevant but constitute signal processing modules that can be combined to create other logical functions and that, in contrast to amplification, cannot be achieved with a single cascade or with two non-interacting cascades. Our computational results revealed surprising similarities between experimental data describing the JNK/MKK4/MKK7 pathway and the solution for

  1. Nobiletin induces inhibitions of Ras activity and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling to suppress cell proliferation in C6 rat glioma cells.

    PubMed

    Aoki, Koichi; Yokosuka, Akihito; Mimaki, Yoshihiro; Fukunaga, Kohji; Yamakuni, Tohru

    2013-01-01

    Ras, a small G-protein, physiologically directs cell proliferation and cell cycle via regulation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling cascade. Dysregulation of Ras/MEK/ERK signaling has been reported to cause tumorigenesis and gliomas. Nobiletin, a citrus flavonoid, has been shown to have anti-tumor cells action. However, it remains elusive whether nobiletin could affect Ras activity. In this study, we provide the first evidence that nobiletin suppresses the proliferation by inhibiting Ras activity in C6 glioma cells, a rat glioma cell line. First, Ras pull-down assay showed that nobiletin inhibits Ras activity in a concentration-dependent manner in C6 cells. Second, farnesyltransferase inhibitor I, a Ras inhibitor, and U0126, a MEK inhibitor, induced an inhibition of the cell proliferation in C6 cells, while the cell proliferation was inhibited by nobiletin as well. Third, western blotting revealed that nobiletin showed inhibitory effects on MEK and ERK phopsphorylation levels in a concentration-dependent manner. Finally, such an inhibitory effect on the level of ERK phosphorylation by nobiletin was appreciably prevented by Gö6976, a selective inhibitor of conventional protein kinase Cs (PKCs) showing Ca(2+)-sensitivity, while GF109203X, a general inhibitor for PKCs, and BAPTA, a cell-permeable Ca(2+) chelator, to a lesser extent, suppressed a reduction of the phosphorylation. These findings suggest that the proliferation of C6 cells is Ras- and MEK/ERK signaling-dependent, and that nobiletin suppresses the cell proliferation by inhibiting Ras activity and MEK/ERK signaling cascade probably via a Ca(2+)-sensitive PKC-dependent mechanism. Thus, the natural compound has potential to be a therapeutic agent for glioma.

  2. Phosphorylation of the fused protein kinase in response to signaling from hedgehog.

    PubMed Central

    Thérond, P P; Knight, J D; Kornberg, T B; Bishop, J M

    1996-01-01

    The hedgehog gene (hh) of Drosophila melanogaster exerts both short- and long-range effects on cell patterning during development. The product of hedgehog is a secreted protein that apparently acts by triggering an intra-cellular signaling pathway, but little is known about the details of that pathway. The Drosophila gene fused (fu) encodes a serine/threonine-protein kinase that genetic experiments have implicated in signaling initiated by hedgehog. Here we report that the fused protein is phosphorylated during the course of Drosophila embryogenesis, as a result of hedgehog activity. In cell culture, phosphorylation of fused protein occurs in response to the biologically active form of hedgehog and cannot be blocked by activation of protein kinase A, which is thought to be an antagonist of signaling from hedgehog. These results suggest that fused and protein kinase A function downstream of hedgehog but in parallel pathways that eventually converge distal to fused. The reconstruction of signaling from hedgehog in cell culture should provide further access to the mechanisms by which hedgehog acts. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8633045

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

    PubMed

    Lee, B N; Elion, E A

    1999-10-26

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

  4. 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

  5. Involvement of mitogen-activated protein kinase activation in the signal-transduction pathways of the soya bean oxidative burst.

    PubMed Central

    Taylor, A T; Kim, J; Low, P S

    2001-01-01

    The oxidative burst constitutes one of the most rapid defence responses characterized in the Plant Kingdom. We have observed that four distinct elicitors of the soya bean oxidative burst activate kinases of masses approximately 44 kDa and approximately 47 kDa. Evidence that these kinases regulate production of reactive oxygen species include: (i) their rapid activation by oxidative burst elicitors, (ii) their tight temporal correlation between activation/deactivation of the kinases and activation/deactivation of the oxidative burst, (iii) the identical pharmacological profile of kinase activation and oxidant production for 13 commonly used inhibitors, and (iv) the autologous activation of both kinases and oxidant production by calyculin A and cantharidin, two phosphatase inhibitors. Immunological and biochemical studies reveal that the activated 44 kDa and 47 kDa kinases are mitogen-activated protein (MAP) kinase family members. The kinases prefer myelin basic protein as a substrate, and they phosphorylate primarily on threonine residues. The kinases are themselves phosphorylated on tyrosine residues, and this phosphorylation is required for activity. Finally, both kinases are recognized by an antibody against activated MAP kinase immediately after (but not before) cell stimulation by elicitors. Based on these and other observations, a preliminary sequence of signalling steps linking elicitor stimulation, kinase activation and Ca(2+) entry, to initiation of oxidant production, is proposed. PMID:11311144

  6. 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

  7. 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

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

    PubMed

    Yoo, Sa Kan; Freisinger, Christina M; LeBert, Danny C; Huttenlocher, Anna

    2012-10-15

    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 H(2)O(2) 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 H(2)O(2). 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.

  9. Macrophage signalling upon mycobacterial infection: the MAP kinases lead the way.

    PubMed

    Schorey, Jeffrey S; Cooper, Andrea M

    2003-03-01

    Mycobacteria activate a series of macrophage signalling pathways upon engaging host cell receptors and during the invasion process. These signals initiate a cascade of events leading to the production of immune effector molecules including cytokines, chemokines and reactive nitrogen intermediates. This response by the macrophage is critical for the control of the mycobacterial infection and, not surprisingly, pathogenic mycobacteria have evolved mechanisms to limit this macrophage activation. Recent data has suggested that macrophages infected with pathogenic compared to non-pathogenic mycobacteria are restricted in their activation of the mitogen activated protein kinase (MAPK) pathways. Mitogen activated protein kinase activation in macrophages appears to play an important role in promoting antimycobacterial activity and in the production of various effector molecules following a mycobacterial infection. Therefore, the ability of pathogenic mycobacteria to limit MAPK activity is likely an important virulence mechanism and may be a potential therapeutic target.

  10. 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

  11. Synaptic generation of an intracellular retrograde signal requires activation of the tyrosine kinase and mitogen-activated protein kinase signaling cascades in Aplysia.

    PubMed

    Stough, Shara; Kopec, Ashley M; Carew, Thomas J

    2015-11-01

    Cellular changes underlying memory formation can be generated in an activity-dependent manner at specific synapses. Thus an important question concerns the mechanisms by which synaptic signals communicate with the cell body to mediate these cellular changes. A monosynaptic circuit that is enhanced by sensitization in Aplysia is well-suited to study this question because three different subcellular compartments: (i) the sensorimotor SN-MN synapses, (ii) the SN projections to MNs via axonal connections, (iii) the SN cell bodies, can all be manipulated and studied independently. Here, we report that activity-dependent (AD) training in either the entire SN-MN circuit or in only the synaptic compartment, activates MAPK in a temporally and spatially specific pattern. Specifically, we find (i) MAPK activation is first transiently generated at SN-MN synapses during training, (ii) immediately after training MAPK is transiently activated in SN-MN axonal connections and persistently activated in SN cell bodies, and finally, (iii) MAPK is activated in SN cell bodies and SN-MN synapses 1h after training. These data suggest that there is an intracellularly transported retrograde signal generated at the synapse which is later responsible for delayed MAPK activation at SN somata. Finally, we find that this retrograde signal requires activation of tyrosine kinase (TK) and MEK signaling cascades at the synapses.

  12. 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

  13. Differential Phosphoproteomics of Fibroblast Growth Factor Signaling: Identification of Src Family Kinase-Mediated Phosphorylation Events

    PubMed Central

    2010-01-01

    Activation of signal transduction by the receptor tyrosine kinase, fibroblast growth factor receptor (FGFR), results in a cascade of protein−protein interactions that rely on the occurrence of specific tyrosine phosphorylation events. One such protein recruited to the activated receptor complex is the nonreceptor tyrosine kinase, Src, which is involved in both initiation and termination of further signaling events. To gain a further understanding of the tyrosine phosphorylation events that occur during FGF signaling, with a specific focus on those that are dependent on Src family kinase (SFK) activity, we have applied SILAC combined with chemical inhibition of SFK activity to search for phosphorylation events that are dependent on SFK activity in FGF stimulated cells. In addition, we used a more targeted approach to carry out high coverage phosphopeptide mapping of one Src substrate protein, the multifunctional adaptor Dok1, and to identify SFK-dependent Dok1 binding partners. From these analyses we identify 80 SFK-dependent phosphorylation events on 40 proteins. We further identify 18 SFK-dependent Dok1 interactions and 9 SFK-dependent Dok1 phosphorylation sites, 6 of which had not previously been known to be SFK-dependent. PMID:20225815

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

    PubMed Central

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

    2015-01-01

    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

  15. Extracellular signals and receptor-like kinases regulating ROP GTPases in plants

    PubMed Central

    Miyawaki, Kaori N.; Yang, Zhenbiao

    2014-01-01

    Rho-like GTPase from plants (ROPs) function as signaling switches that control a wide variety of cellular functions and behaviors including cell morphogenesis, cell division and cell differentiation. The Arabidopsis thaliana genome encodes 11 ROPs that form a distinct single subfamily contrarily to animal or fungal counterparts where multiple subfamilies of Rho GTPases exist. Since Rho proteins bind to their downstream effector proteins only in their GTP-bound “active” state, the activation of ROPs by upstream factor(s) is a critical step in the regulation of ROP signaling. Therefore, it is critical to examine the input signals that lead to the activation of ROPs. Recent findings showed that the plant hormone auxin is an important signal for the activation of ROPs during pavement cell morphogenesis as well as for other developmental processes. In contrast to auxin, another plant hormone, abscisic acid, negatively regulates ROP signaling. Calcium is another emerging signal in the regulation of ROP signaling. Several lines of evidence indicate that plasma membrane localized-receptor like kinases play a critical role in the transmission of the extracellular signals to intracellular ROP signaling pathways. This review focuses on how these signals impinge upon various direct regulators of ROPs to modulate various plant processes. PMID:25295042

  16. 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.

  17. 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.

  18. 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

  19. Regulation of chondrogenesis by protein kinase C: Emerging new roles in calcium signalling.

    PubMed

    Matta, Csaba; Mobasheri, Ali

    2014-05-01

    During chondrogenesis, complex intracellular signalling pathways regulate an intricate series of events including condensation of chondroprogenitor cells and nodule formation followed by chondrogenic differentiation. Reversible phosphorylation of key target proteins is of particular importance during this process. Among protein kinases known to be involved in these pathways, protein kinase C (PKC) subtypes play pivotal roles. However, the precise function of PKC isoenzymes during chondrogenesis and in mature articular chondrocytes is still largely unclear. In this review, we provide a historical overview of how the concept of PKC-mediated chondrogenesis has evolved, starting from the first discoveries of PKC isoform expression and activity. Signalling components upstream and downstream of PKC, leading to the stimulation of chondrogenic differentiation, are also discussed. Although it is evident that we are only at the beginning to understand what roles are assigned to PKC subtypes during chondrogenesis and how they are regulated, there are many yet unexplored aspects in this area. There is evidence that calcium signalling is a central regulator in differentiating chondroprogenitors; still, clear links between intracellular calcium signalling and prototypical calcium-dependent PKC subtypes such as PKCalpha have not been established. Exploiting putative connections and shedding more light on how exactly PKC signalling pathways influence cartilage formation should open new perspectives for a better understanding of healthy as well as pathological differentiation processes of chondrocytes, and may also lead to the development of novel therapeutic approaches. PMID:24440668

  20. 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.

  1. The phosphoproteome of Arabidopsis plants lacking the oxidative signal-inducible1 (OXI1) protein kinase.

    PubMed

    Howden, Andrew J M; Salek, Mogjiborahman; Miguet, Laurent; Pullen, Margaret; Thomas, Benjamin; Knight, Marc R; Sweetlove, Lee J

    2011-04-01

    The AGC protein kinase OXI1 is a key protein in plant responses to oxidative signals, and is important for two oxidative burst-mediated processes: basal resistance to microbial pathogens and root hair growth. To identify possible components of the OXI1 signalling pathway, phosphoproteomic techniques were used to detect alterations in the abundance of phosphorylated proteins and peptides in an oxi1 null mutant of Arabidopsis thaliana. The relative abundance of phosphorylated proteins was assessed either using two-dimensional gel electrophoresis and staining with the phosphoprotein stain Pro-Q Diamond or by the identification and quantification, by mass spectrometry, of stable-isotope labelled phosphopeptides. A number of proteins show altered phosphorylation in the oxi1 mutant. Five proteins, including a putative F-box and 3-phosphoinositide-dependent kinase 1, show reduced phosphorylation in the oxi1 mutant, and may be direct or indirect targets of OXI1. Four proteins, including ethylene insensitive 2 and phospholipase d-gamma, show increased phosphorylation in the oxi1 mutant. This study has identified a range of candidate proteins from the OXI1 signalling pathway. The diverse activities of these proteins, including protein degradation and hormone signalling, may suggest crosstalk between OXI1 and other signal transduction cascades.

  2. 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

  3. 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

  4. C. elegans serine-threonine kinase KIN-29 modulates TGFβ signaling and regulates body size formation

    PubMed Central

    Maduzia, Lisa L; Roberts, Andrew F; Wang, Huang; Lin, Xia; Chin, Lena J; Zimmerman, Cole M; Cohen, Stephen; Feng, Xin-Hua; Padgett, Richard W

    2005-01-01

    Background In C. elegans there are two well-defined TGFβ-like signaling pathways. The Sma/Mab pathway affects body size morphogenesis, male tail development and spicule formation while the Daf pathway regulates entry into and exit out of the dauer state. To identify additional factors that modulate TGFβ signaling in the Sma/Mab pathway, we have undertaken a genetic screen for small animals and have identified kin-29. Results kin-29 encodes a protein with a cytoplasmic serine-threonine kinase and a novel C-terminal domain. The kinase domain is a distantly related member of the EMK (ELKL motif kinase) family, which interacts with microtubules. We show that the serine-threonine kinase domain has in vitro activity. kin-29 mutations result in small animals, but do not affect male tail morphology as do several of the Sma/Mab signal transducers. Adult worms are smaller than the wild-type, but also develop more slowly. Rescue by kin-29 is achieved by expression in neurons or in the hypodermis. Interaction with the dauer pathway is observed in double mutant combinations, which have been seen with Sma/Mab pathway mutants. We show that kin-29 is epistatic to the ligand dbl-1, and lies upstream of the Sma/Mab pathway target gene, lon-1. Conclusion kin-29 is a new modulator of the Sma/Mab pathway. It functions in neurons and in the hypodermis to regulate body size, but does not affect all TGFβ outputs, such as tail morphogenesis. PMID:15840165

  5. Eukaryotic Elongation Factor 2 Kinase Activity Is Controlled by Multiple Inputs from Oncogenic Signaling

    PubMed Central

    Wang, Xuemin; Regufe da Mota, Sergio; Liu, Rui; Moore, Claire E.; Xie, Jianling; Lanucara, Francesco; Agarwala, Usha; Pyr dit Ruys, Sébastien; Vertommen, Didier; Rider, Mark H.; Eyers, Claire E.

    2014-01-01

    Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal α-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that the mTORC1 pathway and the oncogenic Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote from the N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways. PMID:25182533

  6. Mitogen-activated protein kinase phosphatase 1 negatively regulates MAPK signaling in mouse hypothalamus.

    PubMed

    Adachi, Koichi; Goto, Motomitsu; Onoue, Takeshi; Tsunekawa, Taku; Shibata, Miyuki; Hagimoto, Shigeru; Ito, Yoshihiro; Banno, Ryoichi; Suga, Hidetaka; Sugimura, Yoshihisa; Oiso, Yutaka; Arima, Hiroshi

    2014-05-21

    Mitogen-activated protein kinase phosphatase 1 (MKP-1) is shown to negatively regulate MAPK signaling in various peripheral tissues as well as the central nervous system such as cortex, striatum and hippocampus. In this study, we examined whether MKP-1 regulates MAPK signaling in the mouse hypothalamus. Intraperitoneal injection of TNFα significantly increased MKP-1 mRNA expression in paraventricular and arcuate nuclei in the hypothalamus. TNFα treatment induced increases in MKP-1 expression at both mRNA and protein levels, accompanied by the inactivation of MAPK signaling in mouse hypothalamic explants. Inhibition of MKP-1 by its inhibitor or siRNA increased MAPK activity in the explants. Our data indicate that MKP-1 negatively regulates MAPK signaling in the mouse hypothalamus.

  7. 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

  8. 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

  9. Bone Morphogenetic Protein-7 Antagonizes Myocardial Fibrosis Induced by Atrial Fibrillation by Restraining Transforming Growth Factor-β (TGF-β)/Smads Signaling

    PubMed Central

    Chen, Xinjun; Xu, Jing; Jiang, Baozhou; Liu, Danping

    2016-01-01

    Background This aim of this study was to investigate the expression of BMP-7 in atrial fibrillation and illuminate the role of BMP-7 and TGF-β/Smads signaling in myocardial fibrosis. Material/Methods Fibrosis of myocardial fibroblasts was induced by TGF-β1 and the optimal condition was determined by the MTT assay. Cells with TGF-β1 treatment were sub-divided into 4 groups: TGF-β1 group, TGF-β1 + Smad3 siRNA group, TGF-β1 + BMP-7 group, and TGF-β1 + BMP-7 + Smad1/5 siRNA group. Cells were then analyzed by detecting the expression of epithelial cadherin (E-cadherin), collagen I, alpha smooth muscle cell actin (α-SMA), and activated Smads using Western blot. Mice were injected daily with Ach-CaCl2 with or without the addition of BMP-7 and Smad1/5 siRNA over a period of 4 weeks. Cardiac functions were tested by echocardiogram assay and fibrosis was diagnosed by histopathological examination. Finally, molecule biomarkers were detected using standard procedures. Results TGF-β1 treatment significantly down-regulated E-cadherin expression and up-regulated expressions of Collagen I, α-SMA, and pSmad3 (P<0.05). The effects of TGF-β1 treatment can be significantly suppressed by Smad3 siRNA (P<0.05). Cells in the BMP-7 group exhibited significantly higher expression levels of E-cadherin and pSmad1/5 together with lower expression levels of pSmad3, collagen I, and α-SMA (P<0.05). Moreover, Smad1/5 siRNA can substantially repress the effects of BMP-7 (P<0.05) and results from the mice model coincided with those in myocardial fibroblasts. Conclusions BMP-7 can regulate TGF-β1/Smad3 by targeting Smad1/5 to antagonize fibrosis in myocardial fibroblasts resulting from atrial fibrillation. PMID:27677228

  10. Protein-serine/threonine/tyrosine kinases in bacterial signaling and regulation.

    PubMed

    Cousin, Charlotte; Derouiche, Abderahmane; Shi, Lei; Pagot, Yves; Poncet, Sandrine; Mijakovic, Ivan

    2013-09-01

    In this review, we address some recent developments in the field of bacterial protein phosphorylation, focusing specifically on serine/threonine and tyrosine kinases. We present an overview of recent studies outlining the scope of physiological processes that are regulated by phosphorylation, ranging from cell cycle, growth, cell morphology, to metabolism, developmental phenomena, and virulence. Specific emphasis is placed on Mycobacterium tuberculosis as a showcase organism for serine/threonine kinases, and Bacillus subtilis to illustrate the importance of protein phosphorylation in developmental processes. We argue that bacterial serine/threonine and tyrosine kinases have a distinctive feature of phosphorylating multiple substrates and might thus represent integration nodes in the signaling network. Some open questions regarding the evolutionary benefits of relaxed substrate selectivity of these kinases are treated, as well as the notion of nonfunctional 'background' phosphorylation of cellular proteins. We also argue that phosphorylation events for which an immediate regulatory effect is not clearly established should not be dismissed as unimportant, as they may have a role in cross-talk with other post-translational modifications. Finally, recently developed methods for studying protein phosphorylation networks in bacteria are briefly discussed.

  11. 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

  12. Pleiotrophin signaling through anaplastic lymphoma kinase is rate-limiting for glioblastoma growth.

    PubMed

    Powers, Ciaran; Aigner, Achim; Stoica, Gerald E; McDonnell, Kevin; Wellstein, Anton

    2002-04-19

    Glioblastoma multiforme is the most common highly aggressive human brain cancer, and receptor tyrosine kinases have been implicated in the progression of this malignancy. We have recently identified anaplastic lymphoma kinase (ALK) as a tyrosine kinase receptor for pleiotrophin, a secreted growth factor that is highly expressed during embryonic brain development and in tumors of the central nervous system. Here we report on the contribution of pleiotrophin-ALK signaling to glioblastoma growth. We found ALK overexpressed in human glioblastoma relative to normal brain and detected ALK mRNA in glioblastoma cell lines. We reduced the endogenous ALK in glioblastoma cells by ribozyme targeting and demonstrated that this prevents pleiotrophin-stimulated phosphorylation of the anti-apoptotic protein Akt. Furthermore, this depletion of ALK reduced tumor growth of xenografts in athymic nude mice and prolonged survival of the animals because of increased apoptosis in the tumors. These findings directly implicate ALK signaling as a rate-limiting factor in the growth of glioblastoma multiforme and suggest potential utility of therapeutic targeting of ALK.

  13. 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

    2016-01-07

    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.

  14. 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

  15. 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

  16. 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.

  17. Mitogen-activated protein kinase activation in UV-induced signal transduction.

    PubMed

    Bode, Ann M; Dong, Zigang

    2003-01-28

    Experimental evidence supported by epidemiological findings suggests that solar ultraviolet (UV) irradiation is the most important environmental carcinogen leading to the development of skin cancers. Because the ozone layer blocks UVC (wavelength, 180 to 280 nm) exposure, UVA (UVA I, 340 to 400 nm; UVA II, 320 to 340 nm) and UVB (280 to 320 nm) are probably the chief carcinogenic components of sunlight with relevance for human skin cancer. Substantial contributions to the elucidation of the specific signal transduction pathways involved in UV-induced skin carcinogenesis have been made over the past few years, and most evidence suggests that the cellular signaling response is UV wavelength-dependent. The mitogen-activated protein kinase (MAPK) signaling cascades are targets for UV and are important in the regulation of the multitude of UV-induced cellular responses. Experimental studies have used a range of UVA, UVB, UVC, and various combinations in multiple doses, and the observed effects on activation and phosphorylation of MAPKs are varied. This review focuses on the mechanistic data supporting a role for MAPKs in UV-induced skin carcinogenesis. Progress in understanding the mechanisms of UV-induced signal transduction could lead to the use of these protein kinases as specific targets for the prevention and control of skin cancer.

  18. 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

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

    PubMed

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

    2015-07-01

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

  20. Spironolactone Attenuates Experimental Uremic Cardiomyopathy by Antagonizing Marinobufagenin

    PubMed Central

    Tian, Jiang; Shidyak, Amjad; Periyasamy, Sankaridrug M.; Haller, Steven; Taleb, Mohamed; El-Okdi, Nasser; Elkareh, Jihad; Gupta, Shalini; Gohara, Sabry; Fedorova, Olga V.; Cooper, Christopher J.; Xie, Zijian; Malhotra, Deepak; Bagrov, Alexei Y.; Shapiro, Joseph I.

    2009-01-01

    Spironolactone has been noted to attenuate cardiac fibrosis. We have observed that the cardiotonic steroid marinobufagenin plays an important role in the diastolic dysfunction and cardiac fibrosis seen with experimental renal failure. We performed the following studies to determine whether and how spironolactone might ameliorate these changes. First, we studied rats subjected to partial nephrectomy or administration of exogenous marinobufagenin. We found that spironolactone (20 mg/kg per day) attenuated the diastolic dysfunction as assessed by ventricular pressure-volume loops and essentially eliminated cardiac fibrosis as assessed by trichrome staining and Western blot. Next, we examined the effects of spironolactone and its major metabolite, canrenone (both 100 nM), on marinobufagenin stimulation of rat cardiac fibroblasts. Both spironolactone and canrenone prevented the stimulation of collagen production by 1 nM marinobufagenin but not 100 nM marinobufagenin, as assessed by proline incorporation and procollagen 1 expression, as well as signaling through the sodium-potassium-ATPase, as evidenced by protein kinase C isoform δ translocation and extracellular signal regulated kinase 1/2 activation. Both spironolactone and canrenone also altered ouabain binding to cultured porcine cells in a manner consistent with competitive inhibition. Our data suggest that some of the antifibrotic effects of spironolactone may be attributed to antagonism of marinobufagenin signaling through the sodium-potassium-ATPase. PMID:19884563

  1. Spironolactone attenuates experimental uremic cardiomyopathy by antagonizing marinobufagenin.

    PubMed

    Tian, Jiang; Shidyak, Amjad; Periyasamy, Sankaridrug M; Haller, Steven; Taleb, Mohamed; El-Okdi, Nasser; Elkareh, Jihad; Gupta, Shalini; Gohara, Sabry; Fedorova, Olga V; Cooper, Christopher J; Xie, Zijian; Malhotra, Deepak; Bagrov, Alexei Y; Shapiro, Joseph I

    2009-12-01

    Spironolactone has been noted to attenuate cardiac fibrosis. We have observed that the cardiotonic steroid marinobufagenin plays an important role in the diastolic dysfunction and cardiac fibrosis seen with experimental renal failure. We performed the following studies to determine whether and how spironolactone might ameliorate these changes. First, we studied rats subjected to partial nephrectomy or administration of exogenous marinobufagenin. We found that spironolactone (20 mg/kg per day) attenuated the diastolic dysfunction as assessed by ventricular pressure-volume loops and essentially eliminated cardiac fibrosis as assessed by trichrome staining and Western blot. Next, we examined the effects of spironolactone and its major metabolite, canrenone (both 100 nM), on marinobufagenin stimulation of rat cardiac fibroblasts. Both spironolactone and canrenone prevented the stimulation of collagen production by 1 nM marinobufagenin but not 100 nM marinobufagenin, as assessed by proline incorporation and procollagen 1 expression, as well as signaling through the sodium-potassium-ATPase, as evidenced by protein kinase C isoform delta translocation and extracellular signal regulated kinase 1/2 activation. Both spironolactone and canrenone also altered ouabain binding to cultured porcine cells in a manner consistent with competitive inhibition. Our data suggest that some of the antifibrotic effects of spironolactone may be attributed to antagonism of marinobufagenin signaling through the sodium-potassium-ATPase. PMID:19884563

  2. Plasticity of the PAS domain and a potential role for signal transduction in the histidine kinase DcuS.

    PubMed

    Etzkorn, Manuel; Kneuper, Holger; Dünnwald, Pia; Vijayan, Vinesh; Krämer, Jens; Griesinger, Christian; Becker, Stefan; Unden, Gottfried; Baldus, Marc

    2008-10-01

    The mechanistic understanding of how membrane-embedded sensor kinases recognize signals and regulate kinase activity is currently limited. Here we report structure-function relationships of the multidomain membrane sensor kinase DcuS using solid-state NMR, structural modeling and mutagenesis. Experimental data of an individual cytoplasmic Per-Arnt-Sim (PAS) domain were compared to structural models generated in silico. These studies, together with previous NMR work on the periplasmic PAS domain, enabled structural investigations of a membrane-embedded 40-kDa construct by solid-state NMR, comprising both PAS segments and the membrane domain. Structural alterations are largely limited to protein regions close to the transmembrane segment. Data from isolated and multidomain constructs favor a disordered N-terminal helix in the cytoplasmic domain. Mutations of residues in this region strongly influence function, suggesting that protein flexibility is related to signal transduction toward the kinase domain and regulation of kinase activity.

  3. 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

  4. The Yeast Sks1p Kinase Signaling Network Regulates Pseudohyphal Growth and Glucose Response

    PubMed Central

    Johnson, Cole; Kweon, Hye Kyong; Sheidy, Daniel; Shively, Christian A.; Mellacheruvu, Dattatreya; Nesvizhskii, Alexey I.; Andrews, Philip C.; Kumar, Anuj

    2014-01-01

    The yeast Saccharomyces cerevisiae undergoes a dramatic growth transition from its unicellular form to a filamentous state, marked by the formation of pseudohyphal filaments of elongated and connected cells. Yeast pseudohyphal growth is regulated by signaling pathways responsive to reductions in the availability of nitrogen and glucose, but the molecular link between pseudohyphal filamentation and glucose signaling is not fully understood. Here, we identify the glucose-responsive Sks1p kinase as a signaling protein required for pseudohyphal growth induced by nitrogen limitation and coupled nitrogen/glucose limitation. To identify the Sks1p signaling network, we applied mass spectrometry-based quantitative phosphoproteomics, profiling over 900 phosphosites for phosphorylation changes dependent upon Sks1p kinase activity. From this analysis, we report a set of novel phosphorylation sites and highlight Sks1p-dependent phosphorylation in Bud6p, Itr1p, Lrg1p, Npr3p, and Pda1p. In particular, we analyzed the Y309 and S313 phosphosites in the pyruvate dehydrogenase subunit Pda1p; these residues are required for pseudohyphal growth, and Y309A mutants exhibit phenotypes indicative of impaired aerobic respiration and decreased mitochondrial number. Epistasis studies place SKS1 downstream of the G-protein coupled receptor GPR1 and the G-protein RAS2 but upstream of or at the level of cAMP-dependent PKA. The pseudohyphal growth and glucose signaling transcription factors Flo8p, Mss11p, and Rgt1p are required to achieve wild-type SKS1 transcript levels. SKS1 is conserved, and deletion of the SKS1 ortholog SHA3 in the pathogenic fungus Candida albicans results in abnormal colony morphology. Collectively, these results identify Sks1p as an important regulator of filamentation and glucose signaling, with additional relevance towards understanding stress-responsive signaling in C. albicans. PMID:24603354

  5. Scaffold State Switching Amplifies, Accelerates, and Insulates Protein Kinase C Signaling*

    PubMed Central

    Greenwald, Eric C.; Redden, John M.; Dodge-Kafka, Kimberly L.; Saucerman, Jeffrey J.

    2014-01-01

    Scaffold proteins localize two or more signaling enzymes in close proximity to their downstream effectors. A-kinase-anchoring proteins (AKAPs) are a canonical family of scaffold proteins known to bind protein kinase A (PKA) and other enzymes. Several AKAPs have been shown to accelerate, amplify, and specify signal transduction to dynamically regulate numerous cellular processes. However, there is little theory available to mechanistically explain how signaling on protein scaffolds differs from solution biochemistry. In our present study, we propose a novel kinetic mechanism for enzymatic reactions on protein scaffolds to explain these phenomena, wherein the enzyme-substrate-scaffold complex undergoes stochastic state switching to reach an active state. This model predicted anchored enzymatic reactions to be accelerated, amplified, and insulated from inhibition compared with those occurring in solution. We exploited a direct interaction between protein kinase C (PKC) and AKAP7α as a model to validate these predictions experimentally. Using a genetically encoded PKC activity reporter, we found that both the strength and speed of substrate phosphorylation were enhanced by AKAP7α. PKC tethered to AKAP7α was less susceptible to inhibition from the ATP-competitive inhibitor Gö6976 and the substrate-competitive inhibitor PKC 20-28, but not the activation-competitive inhibitor calphostin C. Model predictions and experimental validation demonstrated that insulation is a general property of scaffold tethering. Sensitivity analysis indicated that these findings may be applicable to many other scaffolds as well. Collectively, our findings provide theoretical and experimental evidence that scaffold proteins can amplify, accelerate, and insulate signal transduction. PMID:24302730

  6. Molecular Profiles of Mitogen Activated Protein Kinase Signaling Pathways in Orofacial Development

    PubMed Central

    Singh, Saurabh; Yin, Xiaolong; Pisano, M. Michele; Greene, Robert M.

    2010-01-01

    Background Formation of the mammalian orofacial region involves multiple signaling pathways regulating sequential expression of and interaction between molecular signals during embryogenesis. The present study examined the expression patterns of members of the MAPK family in developing murine orofacial tissue. Methods Total RNA was extracted from developing embryonic orofacial tissue during gestational days (GDs) 12–14 and used to prepare biotinylated cDNA probes, which were then denatured and hybridized to murine MAP kinase signaling pathways gene arrays. Results Expression of a number of genes involved in the (ERK1/2) cascade transiently increased in the embryonic orofacial tissue over the developmental period examined. Numerous members of the SAPK/JNK cascade were constitutively expressed in the tissue. Genes known to play a role in p38 MAPK signaling exhibited constitutive expression during orofacial development. Western blot analysis demonstrated that ERK2/1, p38 and SAPK/JNK kinases are present in embryonic orofacial tissue on each of GD 12, 13 and 14. By using phospho-specific antibodies, active ERK was shown to be temporally regulated during orofacial development. Minimal amounts of active p38 and active SAPK/JNK were detected in orofacial tissue during GDs 12–14. Conclusions Our study documents specific expression patterns of genes coding for proteins belonging to the ERK1/2, p38 and SAPK/JNK MAP kinase families in embryonic orofacial tissue. We also demonstrate that active, phosphorylated forms of ERK1/2 only, were detected in the embryonic tissue investigated, suggesting a more central role for members of this family in embryonic orofacial development. PMID:17177285

  7. Extracellular Signal-Regulated Kinase-2 within the Ventral Tegmental Area Regulates Responses to Stress

    PubMed Central

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

    2010-01-01

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

  8. Mitogen-activated protein kinase cascades in signaling plant growth and development.

    PubMed

    Xu, Juan; Zhang, Shuqun

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are ubiquitous signaling modules in eukaryotes. Early research of plant MAPKs has been focused on their functions in immunity and stress responses. Recent studies reveal that they also play essential roles in plant growth and development downstream of receptor-like protein kinases (RLKs). With only a limited number of MAPK components, multiple functional pathways initiated from different receptors often share the same MAPK components or even a complete MAPK cascade. In this review, we discuss how MAPK cascades function as molecular switches in response to spatiotemporal-specific ligand-receptor interactions and the availability of downstream substrates. In addition, we discuss other possible mechanisms governing the functional specificity of plant MAPK cascades, a question central to our understanding of MAPK functions.

  9. Anaplastic lymphoma kinase and its signalling molecules as novel targets in lymphoma therapy.

    PubMed

    Coluccia, A M L; Gunby, R H; Tartari, C J; Scapozza, L; Gambacorti-Passerini, C; Passoni, Lorena

    2005-06-01

    A crucial issue in the development of molecularly-targeted anticancer therapies is the identification of appropriate molecules whose targeting would result in tumour regression with a minimal level of systemic toxicity. Anaplastic lymphoma kinase (ALK) is a transmembrane receptor tyrosine kinase, normally expressed at low levels in the nervous system. As a consequence of chromosomal translocations involving the alk gene (2p23), ALK is also aberrantly expressed and constitutively activated in approximately 60% of CD30+ anaplastic large cell lymphomas (ALCLs). Due to the selective overexpression of ALK in tumour cells, its direct involvement in the process of malignant transformation and its frequent expression in ALCL patients, the authors recognise ALK as a suitable candidate for the development of molecularly targeted strategies for the therapeutic treatment of ALK-positive lymphomas. Strategies targeting ALK directly or indirectly via the inhibition of the protein networks responsible for ALK oncogenic signalling are discussed. PMID:15948671

  10. Phospho-kinase profile of triple negative breast cancer and androgen receptor signaling

    PubMed Central

    2014-01-01

    Background The androgen receptor (AR) plays a central role in the oncogenesis of different tumors, as is the case in prostate cancer. In triple negative breast cancer (TNBC) a gene expression classification has described different subgroups including a luminal androgen subtype. The AR can be controlled by several mechanisms like the activation of membrane tyrosine kinases and downstream signaling pathways. However little is known in TNBC about how the AR is modulated by these mechanisms and the potential therapeutic strategists to inhibit its expression. Methods We used human samples to evaluate the expression of AR by western-blot and phospho-proteomic kinase arrays that recognize membrane tyrosine kinase receptors and downstream mediators. Western-blots in human cell lines were carried out to analyze the expression and activation of individual proteins. Drugs against these kinases in different conditions were used to measure the expression of the androgen receptor. PCR experiments were performed to assess changes in the AR gene after therapeutic modulation of these pathways. Results AR is present in a subset of TNBC and its expression correlates with activated membrane receptor kinases-EGFR and PDGFRβ in human samples and cell lines. Inhibition of the PI3K/mTOR pathway in TNBC cell lines decreased notably the expression of the AR. Concomitant administration of the anti-androgen bicalutamide with the EGFR, PDGFRβ and Erk1/2 inhibitors, decreased the amount of AR compared to each agent given alone, and had an additive anti-proliferative effect. Administration of dihydrotestosterone augmented the expression of AR that was not modified by the inhibition of the PI3K/mTOR or Erk1/2 pathways. AR expression was posttranscriptionally regulated by PI3K or Erk1/2 inhibition. Conclusion Our results describe the expression of the AR in TNBC as a druggable target and further suggest the combination of bicalutamide with inhibitors of EGFR, PDGFRβ or Erk1/2 for future

  11. Intestinal cell kinase is a novel participant in intestinal cell signaling responses to protein malnutrition.

    PubMed

    Bolick, David T; Chen, Tufeng; Alves, Luís Antonio O; Tong, Yixin; Wu, Di; Joyner, Linwood T; Oriá, Reinaldo B; Guerrant, Richard L; Fu, Zheng

    2014-01-01

    Nutritional deficiency and stress can severely impair intestinal architecture, integrity and host immune defense, leading to increased susceptibility to infection and cancer. Although the intestine has an inherent capability to adapt to environmental stress, the molecular mechanisms by which the intestine senses and responds to malnutrition are not completely understood. We hereby report that intestinal cell kinase (ICK), a highly conserved serine/threonine protein kinase, is a novel component of the adaptive cell signaling responses to protein malnutrition in murine small intestine. Using an experimental mouse model, we demonstrated that intestinal ICK protein level was markedly and transiently elevated upon protein deprivation, concomitant with activation of prominent pro-proliferation and pro-survival pathways of Wnt/β-catenin, mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK), and protein kinase B (PKB/Akt) as well as increased expression of intestinal stem cell markers. Using the human ileocecal epithelial cell line HCT-8 as an in vitro model, we further demonstrated that serum starvation was able to induce up-regulation of ICK protein in intestinal epithelial cells in a reversible manner, and that serum albumin partially contributed to this effect. Knockdown of ICK expression in HCT-8 cells significantly impaired cell proliferation and down-regulated active β-catenin signal. Furthermore, reduced ICK expression in HCT-8 cells induced apoptosis through a caspase-dependent mechanism. Taken together, our findings suggest that increased ICK expression/activity in response to protein deprivation likely provides a novel protective mechanism to limit apoptosis and support compensatory mucosal growth under nutritional stress.

  12. Human cervical cancer cells use Ca2+ signalling, protein tyrosine phosphorylation and MAP kinase in regulatory volume decrease.

    PubMed

    Shen, M R; Chou, C Y; Browning, J A; Wilkins, R J; Ellory, J C

    2001-12-01

    1. This study was aimed at identifying the signalling pathways involved in the activation of volume-regulatory mechanisms of human cervical cancer cells. 2. Osmotic swelling of human cervical cancer cells induced a substantial increase in intracellular Ca2+ ([Ca2+]i) by the activation of Ca2+ entry across the cell membrane, as well as Ca2+ release from intracellular stores. This Ca2+ signalling was critical for the normal regulatory volume decrease (RVD) response. 3. The activation of swelling-activated ion and taurine transport was significantly inhibited by tyrosine kinase inhibitors (genistein and tyrphostin AG 1478) and potentiated by the tyrosine phosphatase inhibitor Na3VO4. However, the Src family of tyrosine kinases was not involved in regulation of the swelling-activated Cl- channel. 4. Cell swelling triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) and p38 kinase. The volume-responsive ERK1/ERK2 signalling pathway linked with the activation of K+ and Cl- channels, and taurine transport. However, the volume-regulatory mechanism was independent of the activation of p38 MAP kinase. 5. The phosphorylated ERK1/ERK2 expression following a hypotonic shock was up-regulated by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and down-regulated by PKC inhibitor staurosporine. The response of ERK activation to hypotonicity also required Ca2+ entry and depended on tyrosine kinase and mitogen-activated/ERK-activating kinase (MEK) activity. 6. Considering the results overall, osmotic swelling promotes the activation of tyrosine kinase and ERK1/ERK2 and raises intracellular Ca2+, all of which play a crucial role in the volume-regulatory mechanism of human cervical cancer cells. PMID:11731569

  13. Brassinosteroid Signal Transduction: From Receptor Kinase Activation to Transcriptional Networks Regulating Plant Development REVIEW

    PubMed Central

    Clouse, Steven D.

    2011-01-01

    Brassinosteroid (BR) signal transduction research has progressed rapidly from the initial discovery of the BR receptor to a complete definition of the basic molecular components required to relay the BR signal from perception by receptor kinases at the cell surface to activation of a small family of transcription factors that regulate the expression of more than a thousand genes in a BR-dependent manner. These mechanistic advances have helped answer the intriguing question of how a single molecule, such as a hormone, can have dramatic pleiotropic effects on a broad range of diverse developmental pathways and have shed light on how BRs interact with other plant hormones and environmental cues to shape the growth of the whole plant. This review summarizes the current state of BR signal transduction research and then examines recent articles uncovering gene regulatory networks through which BR influences both vegetative and reproductive development. PMID:21505068

  14. Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction.

    PubMed

    Davidson, Gary; Wu, Wei; Shen, Jinlong; Bilic, Josipa; Fenger, Ursula; Stannek, Peter; Glinka, Andrei; Niehrs, Christof

    2005-12-01

    Signalling by Wnt proteins (Wingless in Drosophila) has diverse roles during embryonic development and in adults, and is implicated in human diseases, including cancer. LDL-receptor-related proteins 5 and 6 (LRP5 and LRP6; Arrow in Drosophila) are key receptors required for transmission of Wnt/beta-catenin signalling in metazoa. Although the role of these receptors in Wnt signalling is well established, their coupling with the cytoplasmic signalling apparatus remains poorly defined. Using a protein modification screen for regulators of LRP6, we describe the identification of Xenopus Casein kinase 1 gamma (CK1gamma), a membrane-bound member of the CK1 family. Gain-of-function and loss-of-function experiments show that CK1gamma is both necessary and sufficient to transduce LRP6 signalling in vertebrates and Drosophila cells. In Xenopus embryos, CK1gamma is required during anterio-posterior patterning to promote posteriorizing Wnt/beta-catenin signalling. CK1gamma is associated with LRP6, which has multiple, modular CK1 phosphorylation sites. Wnt treatment induces the rapid CK1gamma-mediated phosphorylation of these sites within LRP6, which, in turn, promotes the recruitment of the scaffold protein Axin. Our results reveal an evolutionarily conserved mechanism that couples Wnt receptor activation to the cytoplasmic signal transduction apparatus. PMID:16341016

  15. Expression and localization of the diacylglycerol kinase family and of phosphoinositide signaling molecules in adrenal gland.

    PubMed

    Hozumi, Yasukazu; Akimoto, Ryo; Suzuki, Akihito; Otani, Koichi; Watanabe, Masahiko; Goto, Kaoru

    2015-11-01

    Adrenal glands play a central role in the secretion of steroid hormones and catecholamines. Previous studies have revealed that molecules engaged in phosphoinositide (PI) turnover are expressed in the adrenal gland, suggesting the importance of PI signaling in adrenal signal transduction. Diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol (DG), a major second messenger in the PI signaling cascade. The DGK family is expressed in distinct patterns in endocrine organs at the mRNA and protein levels. Nevertheless, little is known about the characteristics and morphological aspects of DGKs in the adrenal gland. We have performed immunohistochemical analyses to investigate the expression and localization of DGK isozymes, together with PI signaling molecules, in the adrenal gland at the protein level. Our results show that the DGK family and a set of PI signaling molecules are expressed intensely in zona glomerulosa cells and medullary chromaffin cells in the adrenal gland. In adrenal cells, DGKγ localizes to the Golgi complex, DGKε to the plasma membrane, and DGKζ to the nucleus. These findings show the distinct expression and subcellular localization of DGK isozymes and PI signaling molecules in the adrenal gland, suggesting that each DGK isozyme has a role in signal transduction in adrenal cells, especially in the zona glomerulosa and medulla.

  16. NPM-ALK signals through glycogen synthase kinase 3β to promote oncogenesis.

    PubMed

    McDonnell, S R P; Hwang, S R; Basrur, V; Conlon, K P; Fermin, D; Wey, E; Murga-Zamalloa, C; Zeng, Z; Zu, Y; Elenitoba-Johnson, K S J; Lim, M S

    2012-08-01

    Anaplastic large cell lymphoma (ALCL) is the most common type of pediatric peripheral T-cell lymphoma. In 70-80% of cases, the chromosomal aberration t(2;5)(p23;q35) results in the juxtaposition of anaplastic lymphoma kinase (ALK) with nucleophosmin (NPM) and the subsequent expression of the NPM-ALK fusion protein. NPM-ALK is a chimeric tyrosine kinase, which induces numerous signaling pathways that drive proliferation and abrogate apoptosis. However, the mechanisms that lead to activation of downstream growth regulatory molecules have not been completely elucidated. Using a mass spectrometry-based phosphoproteomic screen, we identified GSK3β as a signaling mediator of NPM-ALK. Using a selective inhibitor of ALK, we demonstrated that the tyrosine kinase activity of ALK regulates the serine-9 phosphorylation of GSK3β. Expression of NPM-ALK in 293T cells led to an increase of pS(9)-GSK3β (glycogen synthase kinase 3 beta) compared with kinase-defective K210R mutant NPM-ALK, but did not affect total GSK3β levels. Phosphorylation of pS(9)-GSK3β by NPM-ALK was mediated by the PI3K/AKT signaling pathway. ALK inhibition resulted in degradation of GSK3β substrates Mcl-1 and CDC25A, which was recovered upon chemical inhibition of the proteasome (MG132). Furthermore, the degradation of Mcl-1 was recoverable with inhibition of GSK3β. ALK inhibition also resulted in decreased cell viability, which was rescued by GSK3β inhibition. Furthermore, stable knockdown of GSK3β conferred resistance to the growth inhibitory effects of ALK inhibition using viability and colony formation assays. pS(9)-GSK3β and CDC25A were selectively expressed in neoplastic cells of ALK+ALCL tissue biopsies, and showed a significant correlation (P<0.001). Conversely, ALK-ALCL tissue biopsies did not show significant correlation of pS(9)-GSK3β and CDC25A expression (P<0.2). Our results demonstrate that NPM-ALK regulates the phosphorylation of S(9)-GSK3β by PI3K/AKT. The subsequent inhibition of

  17. Competing G protein-coupled receptor kinases balance G protein and β-arrestin signaling.

    PubMed

    Heitzler, Domitille; Durand, Guillaume; Gallay, Nathalie; Rizk, Aurélien; Ahn, Seungkirl; Kim, Jihee; Violin, Jonathan D; Dupuy, Laurence; Gauthier, Christophe; Piketty, Vincent; Crépieux, Pascale; Poupon, Anne; Clément, Frédérique; Fages, François; Lefkowitz, Robert J; Reiter, Eric

    2012-01-01

    Seven-transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through β-arrestins, whose recruitment to the activated receptor is regulated by G protein-coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal-regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT(1A)R) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)-based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well-established function in the desensitization of G-protein activation, GRK2 exerts a strong negative effect on β-arrestin-dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2-dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT(1A)R, and HEK293 cells expressing other 7TMRs.

  18. Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion

    PubMed Central

    Alam, Mahmood M.; Solyakov, Lev; Bottrill, Andrew R.; Flueck, Christian; Siddiqui, Faiza A.; Singh, Shailja; Mistry, Sharad; Viskaduraki, Maria; Lee, Kate; Hopp, Christine S.; Chitnis, Chetan E.; Doerig, Christian; Moon, Robert W.; Green, Judith L.; Holder, Anthony A.; Baker, David A.; Tobin, Andrew B.

    2015-01-01

    Our understanding of the key phosphorylation-dependent signalling pathways in the human malaria parasite, Plasmodium falciparum, remains rudimentary. Here we address this issue for the essential cGMP-dependent protein kinase, PfPKG. By employing chemical and genetic tools in combination with quantitative global phosphoproteomics, we identify the phosphorylation sites on 69 proteins that are direct or indirect cellular targets for PfPKG. These PfPKG targets include proteins involved in cell signalling, proteolysis, gene regulation, protein export and ion and protein transport, indicating that cGMP/PfPKG acts as a signalling hub that plays a central role in a number of core parasite processes. We also show that PfPKG activity is required for parasite invasion. This correlates with the finding that the calcium-dependent protein kinase, PfCDPK1, is phosphorylated by PfPKG, as are components of the actomyosin complex, providing mechanistic insight into the essential role of PfPKG in parasite egress and invasion. PMID:26149123

  19. Isorhamnetin inhibits cell proliferation and induces apoptosis in breast cancer via Akt and mitogen-activated protein kinase kinase signaling pathways

    PubMed Central

    HU, SHAN; HUANG, LIMING; MENG, LIWEI; SUN, HE; ZHANG, WEI; XU, YINGCHUN

    2015-01-01

    Breast cancer is the most common cause of female cancer-associated mortality. Although treatment options, including chemotherapy, radiotherapy and surgery have led to a decline in the mortality rates associated with breast cancer, drug resistance remains one of the predominant causes for poor prognosis and high recurrence rates. The present study investigated the potential effects of the natural product, isorhamnetin on breast cancer, and examined the effects of isorhamnetin on the Akt/mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinase (MAPK)/MAPK kinase (MEK) signaling cascades, which are two important signaling pathways for endocrine therapy resistance in breast cancer. The results of the present study indicate that isorhamnetin inhibits cell proliferation and induces cell apoptosis. In addition, isorhamnetin was observed to inhibit the Akt/mTOR and the MEK/extracellular signal-regulated kinase phosphorylation cascades. The inhibition of these two signaling pathways was attenuated by the two Akt and MEK1 inhibitors, but not by the nuclear factor-κB inhibitor. Furthermore, epidermal growth factor inhibited the effects of isorhamnetin via activation of the Akt and MEK signaling pathways. These results indicate that isorhamnetin exhibits antitumor effects in breast cancer, which are mediated by the Akt and MEK signaling pathways. PMID:26502751

  20. Isorhamnetin inhibits cell proliferation and induces apoptosis in breast cancer via Akt and mitogen‑activated protein kinase kinase signaling pathways.

    PubMed

    Hu, Shan; Huang, Liming; Meng, Liwei; Sun, He; Zhang, Wei; Xu, Yingchun

    2015-11-01

    Breast cancer is the most common cause of female cancer-associated mortality. Although treatment options, including chemotherapy, radiotherapy and surgery have led to a decline in the mortality rates associated with breast cancer, drug resistance remains one of the predominant causes for poor prognosis and high recurrence rates. The present study investigated the potential effects of the natural product, isorhamnetin on breast cancer, and examined the effects of isorhamnetin on the Akt/mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinase (MAPK)/MAPK kinase (MEK) signaling cascades, which are two important signaling pathways for endocrine therapy resistance in breast cancer. The results of the present study indicate that isorhamnetin inhibits cell proliferation and induces cell apoptosis. In addition, isorhamnetin was observed to inhibit the Akt/mTOR and the MEK/extracellular signal-regulated kinase phosphorylation cascades. The inhibition of these two signaling pathways was attenuated by the two Akt and MEK1 inhibitors, but not by the nuclear factor-κB inhibitor. Furthermore, epidermal growth factor inhibited the effects of isorhamnetin via activation of the Akt and MEK signaling pathways. These results indicate that isorhamnetin exhibits antitumor effects in breast cancer, which are mediated by the Akt and MEK signaling pathways. PMID:26502751

  1. MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway.

    PubMed Central

    Raingeaud, J; Whitmarsh, A J; Barrett, T; Dérijard, B; Davis, R J

    1996-01-01

    The p38 mitogen-activated protein (MAP) kinase signal transduction pathway is activated by proinflammatory cytokines and environmental stress. The detection of p38 MAP kinase in the nucleus of activated cells suggests that p38 MAP kinase can mediate signaling to the nucleus. To test this hypothesis, we constructed expression vectors for activated MKK3 and MKK6, two MAP kinase kinases that phosphorylate and activate p38 MAP kinase. Expression of activated MKK3 and MKK6 in cultured cells caused a selective increase in p38 MAP kinase activity. Cotransfection experiments demonstrated that p38 MAP kinase activation causes increased reporter gene expression mediated by the transcription factors ATF2 and Elk-1. These data demonstrate that the nucleus is one target of the p38 MAP kinase signal transduction pathway. PMID:8622669

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  3. Phosphoproteomic analysis of protein kinase C signaling in Saccharomyces cerevisiae reveals Slt2 mitogen-activated protein kinase (MAPK)-dependent phosphorylation of eisosome core components.

    PubMed

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

    2013-03-01

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

  4. The noble gas argon modifies extracellular signal-regulated kinase 1/2 signaling in neurons and glial cells.

    PubMed

    Fahlenkamp, Astrid V; Rossaint, Rolf; Haase, Hajo; Al Kassam, Hussam; Ryang, Yu-Mi; Beyer, Cordian; Coburn, Mark

    2012-01-15

    Recently, the noble gas argon has been identified as a potent neuroprotective agent, but little is known about its cellular effects. In this in vitro study, we investigated argon's influence on the extracellular signal-regulated kinase (ERK) 1/2, a ubiquitous enzyme with numerous functions in cell proliferation and survival. Primary neuronal and astroglial cell cultures and the microglial cell line BV-2 were exposed to 50 vol.% argon. Further possible effects were studied following stimulation of microglia with 50 ng/ml LPS. ERK 1/2 activation was assessed by phosphorylation state-specific western blotting, cytokine levels by real-time PCR and western blotting. Total phosphotyrosine phosphatase activity was examined with p-nitrophenylphosphate. After 30 min exposure, argon significantly activated ERK 1/2 signaling in microglia. Enhanced phosphorylation of ERK 1/2 was also found in astrocytes and neurons following argon exposure, but it lacked statistical significance. In microglia, argon did not substantially interfere with LPS-induced ERK1/2 activation and inflammatory cytokine induction. Addition of the MEK-Inhibitor U0126 abolished the induced ERK 1/2 phosphorylation. Cellular phosphatase activity and the inactivation of phosphorylated ERK 1/2 were not altered by argon. In conclusion, argon enhanced ERK 1/2 activity in microglia via the upstream kinase MEK, probably through a direct mode of activation. ERK 1/2 signaling in astrocytes and neurons in vitro was also influenced, although not with statistical significance. Whether ERK 1/2 activation by argon affects cellular functions like differentiation and survival in the brain in vivo will have to be determined in future experiments.

  5. Sorafenib inhibits cancer side population cells by targeting c‑Jun N‑terminal kinase signaling.

    PubMed

    Kim, Jong Bin; Lee, Minjong; Park, Seo-Young; Lee, Seulki; Kim, Hye Ri; Lee, Hyo-Suk; Yoon, Jung-Hwan; Kim, Yoon Jun

    2015-12-01

    Sorafenib is a systemic chemotherapeutic agent for advanced hepatocellular carcinoma (HCC). The aim of the present study was to evaluate the anticancer effect of sorafenib in cancer stem cell‑like cells, such as side population (SP) cells, in HCC and to analyze the signaling pathway for drug‑resistance. To evaluate the anticancer effects of sorafenib, Huh7 and Huh‑BAT cells were treated with sorafenib, fluorouracil (5‑FU), and sorafenib plus 5‑FU. These cells were examined for growth rates, the SP fraction, sphere‑forming efficacy and expression of c‑Jun N‑terminal kinase (JNK) signaling molecules. Sorafenib and 5‑FU treatment decreased growth rates in Huh7 and Huh‑BAT cells; however, the treatments exerted different effects in SP cells and on the expression levels of JNK signaling molecules. Treatment with 5‑FU increased the SP cell number and upregulated the expression of JNK signaling molecules. By contrast, sorafenib decreased the SP cell number and downregulated the expression of JNK signaling molecules. No significant differences in sphere‑forming efficacy were observed subsequent to 5‑FU and sorafenib treatment in Huh7 and Huh‑BAT cells. These results indicate that sorafenib exerted anticancer effects in HCC and SP cells by targeting JNK signaling. PMID:26460271

  6. Energetics of Src homology domain interactions in receptor tyrosine kinase-mediated signaling.

    PubMed

    Ladbury, John E; Arold, Stefan T

    2011-01-01

    Intracellular signaling from receptor tyrosine kinases (RTK) on extracellular stimulation is fundamental to all cellular processes. The protein-protein interactions which form the basis of this signaling are mediated through a limited number of polypeptide domains. For signal transduction without corruption, based on a model where signaling pathways are considered as linear bimolecular relays, these interactions have to be highly specific. This is particularly the case when one considers that any cell may have copies of similar binding domains found in numerous proteins. In this work, an overview of the thermodynamics of binding of two of the most common of these domains (SH2 and SH3 domains) is given. This, coupled with insight from high-resolution structural detail, provides a comprehensive survey of how recognition of cognate binding sites for these domains occurs. Based on the data presented, we conclude that specificity offered by these interactions of SH2 and SH3 domains is limited and not sufficient to enforce mutual exclusivity in RTK-mediated signaling. This may explain the current lack of success in pharmaceutical intervention to inhibit the interactions of these domains when they are responsible for aberrant signaling and the resulting disease states such as cancer.

  7. The receptor kinase CERK1 has dual functions in symbiosis and immunity signalling.

    PubMed

    Zhang, Xiaowei; Dong, Wentao; Sun, Jongho; Feng, Feng; Deng, Yiwen; He, Zuhua; Oldroyd, Giles E D; Wang, Ertao

    2015-01-01

    The establishment of symbiotic interactions between mycorrhizal fungi, rhizobial bacteria and their legume hosts involves a common symbiosis signalling pathway. This signalling pathway is activated by Nod factors produced by rhizobia and these are recognised by the Nod factor receptors NFR1/LYK3 and NFR5/NFP. Mycorrhizal fungi produce lipochitooligosaccharides (LCOs) similar to Nod factors, as well as short-chain chitin oligomers (CO4/5), implying commonalities in signalling during mycorrhizal and rhizobial associations. Here we show that NFR1/LYK3, but not NFR5/NFP, is required for the establishment of the mycorrhizal interaction in legumes. NFR1/LYK3 is necessary for the recognition of mycorrhizal fungi and the activation of the symbiosis signalling pathway leading to induction of calcium oscillations and gene expression. Chitin oligosaccharides also act as microbe associated molecular patterns that promote plant immunity via similar LysM receptor-like kinases. CERK1 in rice has the highest homology to NFR1 and we show that this gene is also necessary for the establishment of the mycorrhizal interaction as well as for resistance to the rice blast fungus. Our results demonstrate that NFR1/LYK3/OsCERK1 represents a common receptor for chitooligosaccharide-based signals produced by mycorrhizal fungi, rhizobial bacteria (in legumes) and fungal pathogens. It would appear that mycorrhizal recognition has been conserved in multiple receptors across plant species, but additional diversification in certain plant species has defined other signals that this class of receptors can perceive.

  8. Differential regulation of extracellular signal-regulated protein kinase 1 and Jun N-terminal kinase 1 by Ca2+ and protein kinase C in endothelin-stimulated Rat-1 cells.

    PubMed Central

    Cadwallader, K; Beltman, J; McCormick, F; Cook, S

    1997-01-01

    The extracellular signal-regulated protein kinase (ERK) and Jun N-terminal kinase (JNK) signalling cascades transduce signals from the cell cytoplasm to the nucleus, where they regulate gene expression. The activation of ERK1 by lysophosphatidic acid (LPA) and endothelin 1 (Et-1) was compared in Rat-1 cells. Both stimulated DNA synthesis to a similar degree but, in contrast with LPA, Et-1 did not stimulate sustained ERK1 activation, a signal that is thought to be important for the proliferation of fibroblasts. Et-1, but not LPA, was able to activate JNK1; pharmacological analysis revealed that the same EtA receptor mediates DNA synthesis, ERK1 and JNK1 activation. However, activation of JNK1 required higher concentrations of Et-1 than was required for stimulation of ERK1 or DNA synthesis. Signalling to ERK1 and JNK1 was partly inhibited by pertussis toxin, suggesting that both pathways are regulated in part by Gi or G0 proteins. Activation of JNK1 by Et-1 lagged behind ERK1 activation but was not dependent on it because PD98059, an inhibitor of mitogen-activated protein kinase (or ERK) kinase, was without effect on JNK1 activation. In contrast with recent studies, activation of protein kinase C (PKC) or Ca2+ fluxes inhibited activation of JNK1 but not ERK1; furthermore inhibition of PKC or sequestration of Ca2+ potentiated JNK1 activation by Et-1 but not by anisomycin, and again had little effect on ERK1 activation. These results demonstrate that the same G-protein-coupled receptor can activate both the ERK and JNK signal pathways but the two kinase cascades seem to be separate, parallel pathways that are differentially regulated by PKC and Ca2+. The results are discussed in terms of the role of ERK and JNK in proliferative signalling. PMID:9032468

  9. Dual role for membrane localization in yeast MAP kinase cascade activation and its contribution to signaling fidelity.

    PubMed

    Lamson, Rachel E; Takahashi, Satoe; Winters, Matthew J; Pryciak, Peter M

    2006-03-21

    Distinct MAP kinase pathways in yeast share several signaling components , including the PAK Ste20 and the MAPKKK Ste11, yet signaling is specific. Mating pheromones trigger an initial step in which Ste20 activates Ste11 , and this requires plasma membrane recruitment of the MAP kinase cascade scaffold protein, Ste5 . Here, we demonstrate an additional role for Ste5 membrane localization. Once Ste11 is activated, signaling through the mating pathway remains minimal but is substantially amplified when Ste5 is recruited to the membrane either by the Gbetagamma dimer or by direct membrane targeting, even to internal membranes. Ste11 signaling is also amplified by Ste5 oligomerization and by a hyperactivating mutation in the Ste7 binding region of Ste5. We suggest a model in which membrane recruitment of Ste5 concentrates its binding partners and thereby amplifies signaling through the kinase cascade. We find similar behavior in the osmotically responsive HOG pathway. Remarkably, while both pheromone and hyperosmotic stimuli amplify signaling from constitutively active Ste11, the resulting signaling output remains pathway specific. These findings suggest a common mode of regulation in which pathway stimuli both initiate and amplify MAP kinase cascade signaling. The regulation of rate-limiting steps that lie after a branchpoint from shared components helps ensure signaling specificity.

  10. Protein Tyrosine Kinase 6 Regulates UVB-Induced Signaling and Tumorigenesis in Mouse Skin.

    PubMed

    Chastkofsky, Michael I; Bie, Wenjun; Ball-Kell, Susan M; He, Yu-Ying; Tyner, Angela L

    2015-10-01

    Protein tyrosine kinase 6 (PTK6, also called BRK) is an intracellular tyrosine kinase expressed in the epithelial linings of the gastrointestinal tract and the skin, where it is expressed in nondividing differentiated cells. We found that PTK6 expression increases in the epidermis following UVB treatment. To evaluate the roles of PTK6 in the skin following UVB-induced damage, we exposed back skin of Ptk6 +/+ and Ptk6 -/- SENCAR mice to incremental doses of UVB for 30 weeks. Wild-type mice were more sensitive to UVB and exhibited increased inflammation and greater activation of signal transducer and activator of transcription-3 (STAT3) than Ptk6-/- mice. Disruption of Ptk6 did not have an impact on proliferation, although PTK6 was expressed and activated in basal epithelial cells in wild-type mice following UVB treatment. However, wild-type mice exhibited shortened tumor latency and increased tumor load compared with Ptk6-/- mice, and STAT3 activation was increased in these tumors. PTK6 activation was detected in UVB-induced tumors, and this correlated with increased activating phosphorylation of focal adhesion kinase (FAK) and breast cancer anti-estrogen resistance 1 (BCAR1). Activation of PTK6 was also detected in human squamous cell carcinomas of the skin. Although PTK6 has roles in normal differentiation, it also contributes to UVB-induced injury and tumorigenesis in vivo. PMID:25938342

  11. SH2B1beta adaptor is a key enhancer of RET tyrosine kinase signaling.

    PubMed

    Donatello, S; Fiorino, A; Degl'Innocenti, D; Alberti, L; Miranda, C; Gorla, L; Bongarzone, I; Rizzetti, M G; Pierotti, M A; Borrello, M G

    2007-10-01

    The RET gene encodes two main isoforms of a receptor tyrosine kinase (RTK) implicated in various human diseases. Activating germ-line point mutations are responsible for multiple endocrine neoplasia type 2-associated medullary thyroid carcinomas, inactivating germ-line mutations for Hirschsprung's disease, while somatic rearrangements (RET/PTCs) are specific to papillary thyroid carcinomas. SH2B1beta, a member of the SH2B adaptors family, and binding partner for several RTKs, has been recently described to interact with proto-RET. Here, we show that both RET isoforms and its oncogenic derivatives bind to SH2B1beta through the SRC homology 2 (SH2) domain and a kinase activity-dependent mechanism. As a result, RET phosphorylates SH2B1beta, which in turn enhances its autophosphorylation, kinase activity, and downstream signaling. RET tyrosine residues 905 and 981 are important determinants for functional binding of the adaptor, as removal of both autophosphorylation sites displaces its recruitment. Binding of SH2B1beta appears to protect RET from dephosphorylation by protein tyrosine phosphatases, and might represent a likely mechanism contributing to its upregulation. Thus, overexpression of SH2B1beta, by enhancing phosphorylation/activation of RET transducers, potentiates the cellular differentiation and the neoplastic transformation thereby induced, and counteracts the action of RET inhibitors. Overall, our results identify SH2B1beta as a key enhancer of RET physiologic and pathologic activities.

  12. Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting Dectin-1/syk kinase signaling

    PubMed Central

    Kyrmizi, Irene; Gresnigt, Mark S.; Akoumianaki, Tonia; Samonis, George; Sidiropoulos, Prodromos; Boumpas, Dimitrios; Netea, Mihai G; van de Veerdonk, Frank L; Kontoyiannis, Dimitrios P; Chamilos, Georgios

    2013-01-01

    Aspergillus fumigatus is the predominant airborne fungal pathogen in immunocompromised patients. Genetic defects in NADPH oxidase (chronic granulomatous disease; CGD) and corticosteroid-induced immunosupression lead to impaired killing of A. fumigatus and unique susceptibility to invasive aspergillosis via incompletely characterized mechanisms. Recent studies link Toll-like receptor activation with phagosome maturation via the engagement of autophagy proteins. Herein, we found that infection of human monocytes with A. fumigatus spores triggered selective recruitment of the autophagy protein LC3 II in phagosomes upon fungal cell wall swelling. This response was induced by surface exposure of immunostimulatory β-glucans and was mediated by activation of the Dectin-1 receptor. LC3 II recruitment in A. fumigatus-phagosomes required syk kinase-dependent production of reactive oxygen species (ROS) and was nearly absent in monocytes of patients with CGD. This pathway was important for control of intracellular fungal growth, as silencing of Atg5 resulted in impaired phagosome maturation and killing of A. fumigatus. In-vivo and ex-vivo administration of corticosteroids blocked LC3 II recruitment in A. fumigatus phagosomes via rapid inhibition of syk kinase phosphorylation and downstream production of ROS. Our studies link Dectin-1/syk kinase signaling with maturation of A. fumigatus phagosomes and uncover a mechanism for development of invasive fungal disease. PMID:23817424

  13. An asymmetry-to-symmetry switch in signal transmission by the histidine kinase receptor for TMAO.

    PubMed

    Moore, Jason O; Hendrickson, Wayne A

    2012-04-01

    The osmoregulator trimethylamine-N-oxide (TMAO), commonplace in aquatic organisms, is used as the terminal electron acceptor for respiration in many bacterial species. The TMAO reductase (Tor) pathway for respiratory catalysis is controlled by a receptor system that comprises the TMAO-binding protein TorT, the sensor histidine kinase TorS, and the response regulator TorR. Here we study the TorS/TorT sensor system to gain mechanistic insight into signaling by histidine kinase receptors. We determined crystal structures for complexes of TorS sensor domains with apo TorT and with TorT (TMAO); we characterized TorS sensor associations with TorT in solution; we analyzed the thermodynamics of TMAO binding to TorT-TorS complexes; and we analyzed in vivo responses to TMAO through the TorT/TorS/TorR system to test structure-inspired hypotheses. TorS-TorT(apo) is an asymmetric 2:2 complex that binds TMAO with negative cooperativity to form a symmetric active kinase.

  14. An Asymmetry-to-Symmetry Switch in Signal Transmission by the Histidine Kinase Receptor for TMAO

    SciTech Connect

    Moore, Jason O.; Hendrickson, Wayne A.

    2012-06-28

    The osmoregulator trimethylamine-N-oxide (TMAO), commonplace in aquatic organisms, is used as the terminal electron acceptor for respiration in many bacterial species. The TMAO reductase (Tor) pathway for respiratory catalysis is controlled by a receptor system that comprises the TMAO-binding protein TorT, the sensor histidine kinase TorS, and the response regulator TorR. Here we study the TorS/TorT sensor system to gain mechanistic insight into signaling by histidine kinase receptors. We determined crystal structures for complexes of TorS sensor domains with apo TorT and with TorT (TMAO); we characterized TorS sensor associations with TorT in solution; we analyzed the thermodynamics of TMAO binding to TorT-TorS complexes; and we analyzed in vivo responses to TMAO through the TorT/TorS/TorR system to test structure-inspired hypotheses. TorS-TorT(apo) is an asymmetric 2:2 complex that binds TMAO with negative cooperativity to form a symmetric active kinase.

  15. Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction

    PubMed Central

    Smith, Ira J.; Godinez, Guillermo L.; Singh, Baljit K.; McCaughey, Kelly M.; Alcantara, Raniel R.; Gururaja, Tarikere; Ho, Melissa S.; Nguyen, Henry N.; Friera, Annabelle M.; White, Kathy A.; McLaughlin, John R.; Hansen, Derek; Romero, Jason M.; Baltgalvis, Kristen A.; Claypool, Mark D.; Li, Wei; Lang, Wayne; Yam, George C.; Gelman, Marina S.; Ding, Rongxian; Yung, Stephanie L.; Creger, Daniel P.; Chen, Yan; Singh, Rajinder; Smuder, Ashley J.; Wiggs, Michael P.; Kwon, Oh-Sung; Sollanek, Kurt J.; Powers, Scott K.; Masuda, Esteban S.; Taylor, Vanessa C.; Payan, Donald G.; Kinoshita, Taisei; Kinsella, Todd M.

    2014-01-01

    Controlled mechanical ventilation (CMV) is associated with the development of diaphragm atrophy and contractile dysfunction, and respiratory muscle weakness is thought to contribute significantly to delayed weaning of patients. Therefore, therapeutic strategies for preventing these processes may have clinical benefit. The aim of the current study was to investigate the role of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in CMV-mediated diaphragm wasting and weakness in rats. CMV-induced diaphragm atrophy and contractile dysfunction coincided with marked increases in STAT3 phosphorylation on both tyrosine 705 (Tyr705) and serine 727 (Ser727). STAT3 activation was accompanied by its translocation into mitochondria within diaphragm muscle and mitochondrial dysfunction. Inhibition of JAK signaling during CMV prevented phosphorylation of both target sites on STAT3, eliminated the accumulation of phosphorylated STAT3 within the mitochondria, and reversed the pathologic alterations in mitochondrial function, reduced oxidative stress in the diaphragm, and maintained normal diaphragm contractility. In addition, JAK inhibition during CMV blunted the activation of key proteolytic pathways in the diaphragm, as well as diaphragm atrophy. These findings implicate JAK/STAT3 signaling in the development of diaphragm muscle atrophy and dysfunction during CMV and suggest that the delayed extubation times associated with CMV can be prevented by inhibition of Janus kinase signaling.—Smith, I. J., Godinez, G. L., Singh, B. K., McCaughey, K. M., Alcantara, R. R., Gururaja, T., Ho, M. S., Nguyen, H. N., Friera, A. M., White, K. A., McLaughlin, J. R., Hansen, D., Romero, J. M., Baltgalvis, K. A., Claypool, M. D., Li, W., Lang, W., Yam, G. C., Gelman, M. S., Ding, R., Yung, S. L., Creger, D. P., Chen, Y., Singh, R., Smuder, A. J., Wiggs, M. P., Kwon, O.-S., Sollanek, K. J., Powers, S. K., Masuda, E. S., Taylor, V. C., Payan, D. G

  16. Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction.

    PubMed

    Smith, Ira J; Godinez, Guillermo L; Singh, Baljit K; McCaughey, Kelly M; Alcantara, Raniel R; Gururaja, Tarikere; Ho, Melissa S; Nguyen, Henry N; Friera, Annabelle M; White, Kathy A; McLaughlin, John R; Hansen, Derek; Romero, Jason M; Baltgalvis, Kristen A; Claypool, Mark D; Li, Wei; Lang, Wayne; Yam, George C; Gelman, Marina S; Ding, Rongxian; Yung, Stephanie L; Creger, Daniel P; Chen, Yan; Singh, Rajinder; Smuder, Ashley J; Wiggs, Michael P; Kwon, Oh-Sung; Sollanek, Kurt J; Powers, Scott K; Masuda, Esteban S; Taylor, Vanessa C; Payan, Donald G; Kinoshita, Taisei; Kinsella, Todd M

    2014-07-01

    Controlled mechanical ventilation (CMV) is associated with the development of diaphragm atrophy and contractile dysfunction, and respiratory muscle weakness is thought to contribute significantly to delayed weaning of patients. Therefore, therapeutic strategies for preventing these processes may have clinical benefit. The aim of the current study was to investigate the role of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in CMV-mediated diaphragm wasting and weakness in rats. CMV-induced diaphragm atrophy and contractile dysfunction coincided with marked increases in STAT3 phosphorylation on both tyrosine 705 (Tyr705) and serine 727 (Ser727). STAT3 activation was accompanied by its translocation into mitochondria within diaphragm muscle and mitochondrial dysfunction. Inhibition of JAK signaling during CMV prevented phosphorylation of both target sites on STAT3, eliminated the accumulation of phosphorylated STAT3 within the mitochondria, and reversed the pathologic alterations in mitochondrial function, reduced oxidative stress in the diaphragm, and maintained normal diaphragm contractility. In addition, JAK inhibition during CMV blunted the activation of key proteolytic pathways in the diaphragm, as well as diaphragm atrophy. These findings implicate JAK/STAT3 signaling in the development of diaphragm muscle atrophy and dysfunction during CMV and suggest that the delayed extubation times associated with CMV can be prevented by inhibition of Janus kinase signaling.-Smith, I. J., Godinez, G. L., Singh, B. K., McCaughey, K. M., Alcantara, R. R., Gururaja, T., Ho, M. S., Nguyen, H. N., Friera, A. M., White, K. A., McLaughlin, J. R., Hansen, D., Romero, J. M., Baltgalvis, K. A., Claypool, M. D., Li, W., Lang, W., Yam, G. C., Gelman, M. S., Ding, R., Yung, S. L., Creger, D. P., Chen, Y., Singh, R., Smuder, A. J., Wiggs, M. P., Kwon, O.-S., Sollanek, K. J., Powers, S. K., Masuda, E. S., Taylor, V. C., Payan, D. G

  17. The Tec kinase-regulated phosphoproteome reveals a mechanism for the regulation of inhibitory signals in murine macrophages

    PubMed Central

    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.

    2015-01-01

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

  18. PfIRR Interacts with HrIGF-I and Activates the MAP-kinase and PI3-kinase Signaling Pathways to Regulate Glycogen Metabolism in Pinctada fucata

    PubMed Central

    Shi, Yu; He, Mao-xian

    2016-01-01

    The insulin-induced mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways are major intracellular signaling modules and conserved among eukaryotes that are known to regulate diverse cellular processes. However, they have not been investigated in the mollusk species Pinctada fucata. Here, we demonstrate that insulin-related peptide receptor of P. fucata (pfIRR) interacts with human recombinant insulin-like growth factor I (hrIGF-I), and stimulates the MAPK and PI3K signaling pathways in P. fucata oocytes. We also show that inhibition of pfIRR by the inhibitor PQ401 significantly attenuates the basal and hrIGF-I-induced phosphorylation of MAPK and PI3K/Akt at amino acid residues threonine 308 and serine 473. Furthermore, our experiments show that there is cross-talk between the MAPK and PI3K/Akt pathways, in which MAPK kinase positively regulates the PI3K pathway, and PI3K positively regulates the MAPK cascade. Intramuscular injection of hrIGF-I stimulates the PI3K and MAPK pathways to increase the expression of pfirr, protein phosphatase 1, glucokinase, and the phosphorylation of glycogen synthase, decreases the mRNA expression of glycogen synthase kinase-3 beta, decreases glucose levels in hemocytes, and increases glycogen levels in digestive glands. These results suggest that the MAPK and PI3K pathways in P. fucata transmit the hrIGF-I signal to regulate glycogen metabolism. PMID:26911653

  19. PfIRR Interacts with HrIGF-I and Activates the MAP-kinase and PI3-kinase Signaling Pathways to Regulate Glycogen Metabolism in Pinctada fucata.

    PubMed

    Shi, Yu; He, Mao-xian

    2016-01-01

    The insulin-induced mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways are major intracellular signaling modules and conserved among eukaryotes that are known to regulate diverse cellular processes. However, they have not been investigated in the mollusk species Pinctada fucata. Here, we demonstrate that insulin-related peptide receptor of P. fucata (pfIRR) interacts with human recombinant insulin-like growth factor I (hrIGF-I), and stimulates the MAPK and PI3K signaling pathways in P. fucata oocytes. We also show that inhibition of pfIRR by the inhibitor PQ401 significantly attenuates the basal and hrIGF-I-induced phosphorylation of MAPK and PI3K/Akt at amino acid residues threonine 308 and serine 473. Furthermore, our experiments show that there is cross-talk between the MAPK and PI3K/Akt pathways, in which MAPK kinase positively regulates the PI3K pathway, and PI3K positively regulates the MAPK cascade. Intramuscular injection of hrIGF-I stimulates the PI3K and MAPK pathways to increase the expression of pfirr, protein phosphatase 1, glucokinase, and the phosphorylation of glycogen synthase, decreases the mRNA expression of glycogen synthase kinase-3 beta, decreases glucose levels in hemocytes, and increases glycogen levels in digestive glands. These results suggest that the MAPK and PI3K pathways in P. fucata transmit the hrIGF-I signal to regulate glycogen metabolism.

  20. Protein serine/threonine kinases in signal transduction for secondary metabolism and morphogenesis in Streptomyces.

    PubMed

    Umeyama, T; Lee, P-C; Horinouchi, S

    2002-08-01

    A number of proteins in the Gram-positive bacterial genus Streptomyces are phosphorylated on their serine/threonine and tyrosine residues in response to developmental phases. AfsR is one of these proteins and acts as a transcriptional factor in both the regulation of secondary metabolism in Streptomyces coelicolor A3(2) and morphological differentiation in Streptomyces griseus. In S. coelicolor A3(2), AfsR is phosphorylated on its serine and threonine residues by more than three protein kinases whose kinase activity is enhanced by means of autophosphorylation on their serine and threonine residues. The degree of autophosphorylation of AfsK is regulated by KbpA which, by binding directly to the kinase domain of AfsK, inhibits its autophosphorylation. Phosphorylation of AfsR enhances its DNA-binding activity and causes it to bind the promoter elements, including -35, of afsS, thus resulting in activation of afsS transcription. ATPase activity of AfsR is essential for this transcriptional activation, probably because the energy available from ATP hydrolysis is required for the isomerization of the closed complex between AfsR and RNA polymerase to a transcriptionally competent open complex. afsS, encoding a 63-amino-acid protein, then activates transcription of actII-ORF4, a pathway-specific transcriptional activator in the actinorhodin biosynthetic gene cluster, in an as yet unknown way. Distribution of the afsK- afsR systems in a wide variety of Streptomyces species and the presence of many phosphorylated proteins in a given Streptomyces strain suggest that the signal transduction via not only two-component regulatory systems but also serine/threonine kinases generally regulates secondary metabolism and morphogenesis in this genus.

  1. Expression of human tyrosine kinase-negative epidermal growth factor receptor amplifies signaling through endogenous murine epidermal growth factor receptor.

    PubMed

    Hack, N; Sue-A-Quan, A; Mills, G B; Skorecki, K L

    1993-12-15

    Recent findings have suggested that certain ligand-dependent responses to EGF may be propagated in a manner that is not dependent on the intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGF-R, Campos-Gonzalez, R., and Glenney, J. R., Jr. (1992) J. Biol. Chem. 267, 14535-14538) or, alternatively, that these responses may occur through the interaction of the human tyrosine kinase-deficient EGF-R with an as yet unidentified kinase (Selva, E., Raden, D. L., and Davis, R. J. (1993) J. Biol. Chem. 268, 2250-2254). These conclusions represent a significant departure from our current understanding of signal transduction by receptor tyrosine kinases. Therefore we examined the effect of expression of tyrosine kinase-negative human EGF receptor in murine NIH-3T3-2.2 cells on the EGF-dependent phosphorylation of mitogen-activated protein (MAP-2) kinase. In parental cells (NIH-3T3-2.2) that express low levels of endogenous murine EGF-R, there was no demonstrable EGF-dependent coupling to MAP-2 kinase. In NIH-3T3-2.2 cells transfected with tyrosine kinase-negative human EGF-R, there was unexpected EGF-dependent phosphorylation of MAP-2 kinase. Analysis of the tyrosine kinase-negative human EGF-R in these cells revealed significant tyrosine phosphorylation of the EGF-R. A low level of endogenous murine EGF-R present in these cells were also phosphorylated on tyrosine residues and displayed autokinase activity. Similar results were obtained using an unrelated cell line (B82L cells), in which EGF-dependent phosphorylation of MAP-2 kinase was previously attributed to signal propagation through a tyrosine kinase-negative human EGF-R (Campos-Gonzalez, R., and Glenney, J. R., Jr. (1992) J. Biol. Chem. 267, 14535-14538). Taken together, these results suggest that the tyrosine kinase-negative human EGF-R are able to amplify the response to activation of low levels of endogenous murine EGF-R, thus leading to EGF-dependent phosphorylation of MAP-2 kinase in cells

  2. Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

    SciTech Connect

    Fukumoto, Yasunori Kuki, Kazumasa; Morii, Mariko; Miura, Takahito; Honda, Takuya; Ishibashi, Kenichi; Hasegawa, Hitomi; Kubota, Sho; Ide, Yudai; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto

    2014-09-26

    Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.

  3. Happyhour, a Ste20 family kinase, implicates EGFR signaling in ethanol-induced behaviors.

    PubMed

    Corl, Ammon B; Berger, Karen H; Ophir-Shohat, Galit; Gesch, Julie; Simms, Jeffrey A; Bartlett, Selena E; Heberlein, Ulrike

    2009-05-29

    The consequences of alcohol use disorders (AUDs) are devastating to individuals and society, yet few treatments are currently available. To identify genes regulating the behavioral effects of ethanol, we conducted a genetic screen in Drosophila and identified a mutant, happyhour (hppy), due to its increased resistance to the sedative effects of ethanol. Hppy protein shows strong homology to mammalian Ste20 family kinases of the GCK-1 subfamily. Genetic and biochemical experiments revealed that the epidermal growth factor (EGF)-signaling pathway regulates ethanol sensitivity in Drosophila and that Hppy functions as an inhibitor of the pathway. Acute pharmacological inhibition of the EGF receptor (EGFR) in adult animals altered acute ethanol sensitivity in both flies and mice and reduced ethanol consumption in a preclinical rat model of alcoholism. Inhibitors of the EGFR or components of its signaling pathway are thus potential pharmacotherapies for AUDs.

  4. Nonreceptor protein tyrosine kinase involvement in signal transduction and immunodeficiency disease.

    PubMed

    Saouaf, S J; Burkhardt, A L; Bolen, J B

    1995-09-01

    The nonreceptor protein tyrosine kinases (PTKs) have been grouped into 10 different enzyme families based on predicted amino acid sequences. As the number of enzymes belonging to the nonreceptor class of PTK is increasing, one challenge is to determine how these various classes of PTKs interact within the cell to promote signal transduction. Herein, the activation of four classes of nonreceptor PTKs is discussed in relation to their interactions with each other as well as with other signaling molecules during the process of lymphocyte surface antigen receptor-mediated activation. Recent findings of nonreceptor PTK loss-of-function mutations in different immunodeficiency diseases has revealed the important contribution of this group of enzymes to lymphocyte development. PMID:7554458

  5. Direct Modulation of Heterotrimeric G Protein-coupled Signaling by a Receptor Kinase Complex.

    PubMed

    Tunc-Ozdemir, Meral; Urano, Daisuke; Jaiswal, Dinesh Kumar; Clouse, Steven D; Jones, Alan M

    2016-07-01

    Plants and some protists have heterotrimeric G protein complexes that activate spontaneously without canonical G protein-coupled receptors (GPCRs). In Arabidopsis, the sole 7-transmembrane regulator of G protein signaling 1 (AtRGS1) modulates the G protein complex by keeping it in the resting state (GDP-bound). However, it remains unknown how a myriad of biological responses is achieved with a single G protein modulator. We propose that in complete contrast to G protein activation in animals, plant leucine-rich repeat receptor-like kinases (LRR RLKs), not GPCRs, provide this discrimination through phosphorylation of AtRGS1 in a ligand-dependent manner. G protein signaling is directly activated by the pathogen-associated molecular pattern flagellin peptide 22 through its LRR RLK, FLS2, and co-receptor BAK1.

  6. The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling

    PubMed Central

    Raja, Erna; Edlund, Karolina; Kahata, Kaoru; Zieba, Agata; Morén, Anita; Watanabe, Yukihide; Voytyuk, Iryna; Botling, Johan; Söderberg, Ola; Micke, Patrick; Pyrowolakis, George; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-01-01

    The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. We demonstrate that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease. PMID:26701726

  7. Targeting PI3 kinase/AKT/mTOR signaling in cancer.

    PubMed

    Sheppard, Karen; Kinross, Kathryn M; Solomon, Benjamin; Pearson, Richard B; Phillips, Wayne A

    2012-01-01

    The phosphatidylinositol 3 kinase (PI3K) pathway is one of the major pathways modulating cell growth, proliferation, metabolism, survival, and angiogenesis. Hyperactivation of this pathway is one of the most frequent occurrences in human cancer and is thus an obvious target for treatment of this disease. Currently there are 26 novel compounds targeting the PI3K pathway being assessed in more than 150 cancer-related clinical trials. Although this pathway is involved in many vital biologic functions, data emanating from these clinical trials indicate that these drugs are well tolerated. This review outlines the interaction of the PI3K pathway with other signaling cascades, highlights mechanisms involved in hyperactivation, discusses current therapeutics in cancer-related clinical trials that target this pathway, and, based on preclinical data, discusses possible leads on patient selection and combinational therapy, including targeting multiple components of the associated signaling network.

  8. Glycogen synthase kinase-3 (GSK3) inhibition induces prosurvival autophagic signals in human pancreatic cancer cells.

    PubMed

    Marchand, Benoît; Arsenault, Dominique; Raymond-Fleury, Alexandre; Boisvert, François-Michel; Boucher, Marie-Josée

    2015-02-27

    Glycogen synthase kinase-3 (GSK3) are ubiquitously expressed serine-threonine kinases involved in a plethora of functions ranging from the control of glycogen metabolism to transcriptional regulation. We recently demonstrated that GSK3 inhibition triggers JNK-cJUN-dependent apoptosis in human pancreatic cancer cells. However, the comprehensive picture of downstream GSK3-regulated pathways/functions remains elusive. Herein, counterbalancing the death signals, we show that GSK3 inhibition induces prosurvival signals through increased activity of the autophagy/lysosomal network. Our data also reveal a contribution of GSK3 in the regulation of the master transcriptional regulator of autophagy and lysosomal biogenesis, transcription factor EB (TFEB) in pancreatic cancer cells. Similarly to mammalian target of rapamycin (mTOR) inhibition, GSK3 inhibitors promote TFEB nuclear localization and leads to TFEB dephosphorylation through endogenous serine/threonine phosphatase action. However, GSK3 and mTOR inhibition impinge differently and independently on TFEB phosphorylation suggesting that TFEB is regulated by a panel of kinases and/or phosphatases. Despite their differential impact on TFEB phosphorylation, both GSK3 and mTOR inhibitors promote 14-3-3 dissociation and TFEB nuclear localization. Quantitative mass spectrometry analyses further reveal an increased association of TFEB with nuclear proteins upon GSK3 and mTOR inhibition suggesting a positive impact on TFEB transcriptional function. Finally, a predominant nuclear localization of TFEB is unveiled in fully fed pancreatic cancer cells, whereas a reduction in TFEB expression significantly impairs their capacity for growth in an anchorage-independent manner. In addition, TFEB-restricted cells are more sensitive to apoptosis upon GSK3 inhibition. Altogether, our data uncover new functions under the control of GSK3 in pancreatic cancer cells in addition to providing key insight into TFEB regulation.

  9. Salt-inducible Kinase 3 Signaling Is Important for the Gluconeogenic Programs in Mouse Hepatocytes.

    PubMed

    Itoh, Yumi; Sanosaka, Masato; Fuchino, Hiroyuki; Yahara, Yasuhito; Kumagai, Ayako; Takemoto, Daisaku; Kagawa, Mai; Doi, Junko; Ohta, Miho; Tsumaki, Noriyuki; Kawahara, Nobuo; Takemori, Hiroshi

    2015-07-17

    Salt-inducible kinases (SIKs), members of the 5'-AMP-activated protein kinase (AMPK) family, are proposed to be important suppressors of gluconeogenic programs in the liver via the phosphorylation-dependent inactivation of the CREB-specific coactivator CRTC2. Although a dramatic phenotype for glucose metabolism has been found in SIK3-KO mice, additional complex phenotypes, dysregulation of bile acids, cholesterol, and fat homeostasis can render it difficult to discuss the hepatic functions of SIK3. The aim of this study was to examine the cell autonomous actions of SIK3 in hepatocytes. To eliminate systemic effects, we prepared primary hepatocytes and screened the small compounds suppressing SIK3 signaling cascades. SIK3-KO primary hepatocytes produced glucose more quickly after treatment with the cAMP agonist forskolin than the WT hepatocytes, which was accompanied by enhanced gluconeogenic gene expression and CRTC2 dephosphorylation. Reporter-based screening identified pterosin B as a SIK3 signaling-specific inhibitor. Pterosin B suppressed SIK3 downstream cascades by up-regulating the phosphorylation levels in the SIK3 C-terminal regulatory domain. When pterosin B promoted glucose production by up-regulating gluconeogenic gene expression in mouse hepatoma AML-12 cells, it decreased the glycogen content and stimulated an association between the glycogen phosphorylase kinase gamma subunit (PHKG2) and SIK3. PHKG2 phosphorylated the peptides with sequences of the C-terminal domain of SIK3. Here we found that the levels of active AMPK were higher both in the SIK3-KO hepatocytes and in pterosin B-treated AML-12 cells than in their controls. These results suggest that SIK3, rather than SIK1, SIK2, or AMPKs, acts as the predominant suppressor in gluconeogenic gene expression in the hepatocytes.

  10. Salt-inducible Kinase 3 Signaling Is Important for the Gluconeogenic Programs in Mouse Hepatocytes*

    PubMed Central

    Itoh, Yumi; Sanosaka, Masato; Fuchino, Hiroyuki; Yahara, Yasuhito; Kumagai, Ayako; Takemoto, Daisaku; Kagawa, Mai; Doi, Junko; Ohta, Miho; Tsumaki, Noriyuki; Kawahara, Nobuo; Takemori, Hiroshi

    2015-01-01

    Salt-inducible kinases (SIKs), members of the 5′-AMP-activated protein kinase (AMPK) family, are proposed to be important suppressors of gluconeogenic programs in the liver via the phosphorylation-dependent inactivation of the CREB-specific coactivator CRTC2. Although a dramatic phenotype for glucose metabolism has been found in SIK3-KO mice, additional complex phenotypes, dysregulation of bile acids, cholesterol, and fat homeostasis can render it difficult to discuss the hepatic functions of SIK3. The aim of this study was to examine the cell autonomous actions of SIK3 in hepatocytes. To eliminate systemic effects, we prepared primary hepatocytes and screened the small compounds suppressing SIK3 signaling cascades. SIK3-KO primary hepatocytes produced glucose more quickly after treatment with the cAMP agonist forskolin than the WT hepatocytes, which was accompanied by enhanced gluconeogenic gene expression and CRTC2 dephosphorylation. Reporter-based screening identified pterosin B as a SIK3 signaling-specific inhibitor. Pterosin B suppressed SIK3 downstream cascades by up-regulating the phosphorylation levels in the SIK3 C-terminal regulatory domain. When pterosin B promoted glucose production by up-regulating gluconeogenic gene expression in mouse hepatoma AML-12 cells, it decreased the glycogen content and stimulated an association between the glycogen phosphorylase kinase gamma subunit (PHKG2) and SIK3. PHKG2 phosphorylated the peptides with sequences of the C-terminal domain of SIK3. Here we found that the levels of active AMPK were higher both in the SIK3-KO hepatocytes and in pterosin B-treated AML-12 cells than in their controls. These results suggest that SIK3, rather than SIK1, SIK2, or AMPKs, acts as the predominant suppressor in gluconeogenic gene expression in the hepatocytes. PMID:26048985

  11. 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

  12. Control of Cardiac Repolarization by Phosphoinositide 3-kinase Signaling to Ion Channels

    PubMed Central

    Ballou, Lisa M.; Lin, Richard Z.; Cohen, Ira S.

    2014-01-01

    Upregulation of phosphoinositide 3-kinase (PI3K) signaling is a common alteration in human cancer, and numerous drugs that target this pathway have been developed for cancer treatment. However, recent studies have implicated inhibition of the PI3K signaling pathway as the cause of a drug-induced long QT syndrome in which alterations in several ion currents contribute to arrhythmogenic drug activity. Surprisingly, some drugs that were thought to induce long QT syndrome by direct block of the rapid delayed rectifier (IKr) also appear to inhibit PI3K signaling, an effect that may contribute to their arrhythmogenicity. The importance of PI3K in regulating cardiac repolarization is underscored by evidence that QT interval prolongation in diabetes also may result from changes in multiple currents due to decreased insulin activation of PI3K in the heart. How PI3K signaling regulates ion channels to control the cardiac action potential is poorly understood. Hence, this review summarizes what is known about the impact of PI3K and its downstream effectors including Akt on sodium, potassium and calcium currents in cardiac myocytes. We also refer to some studies in non-cardiac cells that provide insight into potential mechanisms of ion channel regulation by this signaling pathway in the heart. Drug development and safety could be improved with a better understanding of the mechanisms by which PI3K regulates cardiac ion channels and the extent to which PI3K inhibition contributes to arrhythmogenic susceptibility. PMID:25552692

  13. A mechanism for Src kinase-dependent signaling by non-catalytic receptors

    PubMed Central

    2008-01-01

    A fundamental issue in cell biology is how signals are transmitted across membranes. A variety of transmembrane receptors, including multichain immune recognition receptors, lack catalytic activity and require Src family kinases (SFKs) for signal transduction. However, many receptors only bind and activate SFKs after ligand-induced receptor dimerization. This presents a conundrum: How do SFKs sense the dimerization of receptors to which they are not already bound? Most proposals to resolve this enigma invoke additional players, such as lipid rafts or receptor conformational changes. Here we used simple thermodynamics to show that SFK activation is a natural outcome of clustering of receptors with SFK phosphorylation sites, provided that there is phosphorylation-dependent receptor-SFK association and an SFK bound to one receptor can phosphorylate the second receptor or its associated SFK in a dimer. A simple system of receptor, SFK and an unregulated protein tyrosine phosphatase (PTP) can account for ligand-induced changes in phosphorylation observed in cells. We suggest that a core signaling system comprising a receptor with SFK phosphorylation sites, an SFK and an unregulated PTP provides a robust mechanism for transmembrane signal transduction. Other events that regulate signaling in specific cases may have evolved for fine-tuning of this basic mechanism. PMID:18444664

  14. Cellular Notch responsiveness is defined by phosphoinositide 3-kinase-dependent signals

    PubMed Central

    Mckenzie, Grahame; Ward, George; Stallwood, Yvette; Briend, Emmanuel; Papadia, Sofia; Lennard, Andrew; Turner, Martin; Champion, Brian; Hardingham, Giles E

    2006-01-01

    Background Notch plays a wide-ranging role in controlling cell fate, differentiation and development. The PI3K-Akt pathway is a similarly conserved signalling pathway which regulates processes such as differentiation, proliferation and survival. Mice with disrupted Notch and PI3K signalling show phenotypic similarities during haematopoietic cell development, suggesting functional interaction between these pathways. Results We show that cellular responsiveness to Notch signals depends on the activity of the PI3K-Akt pathway in cells as diverse as CHO cells, primary T-cells and hippocampal neurons. Induction of the endogenous PI3K-Akt pathway in CHO cells (by the insulin pathway), in T-cells (via TCR activation) or in neurons (via TrKB activation) potentiates Notch-dependent responses. We propose that the PI3K-Akt pathway exerts its influence on Notch primarily via inhibition of GSK3-beta, a kinase known to phosphorylate and regulate Notch signals. Conclusion The PI3K-Akt pathway acts as a "gain control" for Notch signal responses. Since physiological levels of intracellular Notch are often low, coincidence with PI3K-activation may be crucial for induction of Notch-dependent responses. PMID:16507111

  15. An electrostatic selection mechanism controls sequential kinase signaling downstream of the T cell receptor

    PubMed Central

    Shah, Neel H; Wang, Qi; Yan, Qingrong; Karandur, Deepti; Kadlecek, Theresa A; Fallahee, Ian R; Russ, William P; Ranganathan, Rama; Weiss, Arthur; Kuriyan, John

    2016-01-01

    The sequence of events that initiates T cell signaling is dictated by the specificities and order of activation of the tyrosine kinases that signal downstream of the T cell receptor. Using a platform that combines exhaustive point-mutagenesis of peptide substrates, bacterial surface-display, cell sorting, and deep sequencing, we have defined the specificities of the first two kinases in this pathway, Lck and ZAP-70, for the T cell receptor ζ chain and the scaffold proteins LAT and SLP-76. We find that ZAP-70 selects its substrates by utilizing an electrostatic mechanism that excludes substrates with positively-charged residues and favors LAT and SLP-76 phosphosites that are surrounded by negatively-charged residues. This mechanism prevents ZAP-70 from phosphorylating its own activation loop, thereby enforcing its strict dependence on Lck for activation. The sequence features in ZAP-70, LAT, and SLP-76 that underlie electrostatic selectivity likely contribute to the specific response of T cells to foreign antigens. DOI: http://dx.doi.org/10.7554/eLife.20105.001 PMID:27700984

  16. Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase Inhibitors

    PubMed Central

    Canning, Peter; Ruan, Qui; Schwerd, Tobias; Hrdinka, Matous; Maki, Jenny L.; Saleh, Danish; Suebsuwong, Chalada; Ray, Soumya; Brennan, Paul E.; Cuny, Gregory D.; Uhlig, Holm H.; Gyrd-Hansen, Mads; Degterev, Alexei; Bullock, Alex N.

    2015-01-01

    Summary RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers. PMID:26320862

  17. Single cell kinase signaling assay using pinched flow coupled droplet microfluidics.

    PubMed

    Ramji, Ramesh; Wang, Ming; Bhagat, Ali Asgar S; Tan Shao Weng, Daniel; Thakor, Nitish V; Teck Lim, Chwee; Chen, Chia-Hung

    2014-05-01

    Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel (5 mm) to separate cell aggregates and to form a uniform cell distribution in a droplet-generating platform that encapsulated single cells with >55% encapsulation efficiency beating Poisson encapsulation statistics. Using this platform and commercially available Sox substrates (8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline), we have demonstrated a high throughput dynamic single cell signaling assay to measure the activity of receptor tyrosine kinases (RTKs) in lung cancer cells triggered by cell surface ligand binding. The phosphorylation of the substrates resulted in fluorescent emission, showing a sigmoidal increase over a 12 h period. The result exhibited a heterogeneous signaling rate in individual cells and showed various levels of drug resistance when treated with the tyrosine kinase inhibitor, gefitinib. PMID:24926389

  18. Single cell kinase signaling assay using pinched flow coupled droplet microfluidics

    PubMed Central

    Ramji, Ramesh; Wang, Ming; Bhagat, Ali Asgar S.; Tan Shao Weng, Daniel; Thakor, Nitish V.; Teck Lim, Chwee; Chen, Chia-Hung

    2014-01-01

    Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel (5 mm) to separate cell aggregates and to form a uniform cell distribution in a droplet-generating platform that encapsulated single cells with >55% encapsulation efficiency beating Poisson encapsulation statistics. Using this platform and commercially available Sox substrates (8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline), we have demonstrated a high throughput dynamic single cell signaling assay to measure the activity of receptor tyrosine kinases (RTKs) in lung cancer cells triggered by cell surface ligand binding. The phosphorylation of the substrates resulted in fluorescent emission, showing a sigmoidal increase over a 12 h period. The result exhibited a heterogeneous signaling rate in individual cells and showed various levels of drug resistance when treated with the tyrosine kinase inhibitor, gefitinib. PMID:24926389

  19. Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium.

    PubMed

    Frey, M R; Clark, J A; Leontieva, O; Uronis, J M; Black, A R; Black, J D

    2000-11-13

    Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G(0). PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCalpha alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt-villus axis revealed that PKCalpha activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit-specific events in situ. Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.

  20. Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase Inhibitors.

    PubMed

    Canning, Peter; Ruan, Qui; Schwerd, Tobias; Hrdinka, Matous; Maki, Jenny L; Saleh, Danish; Suebsuwong, Chalada; Ray, Soumya; Brennan, Paul E; Cuny, Gregory D; Uhlig, Holm H; Gyrd-Hansen, Mads; Degterev, Alexei; Bullock, Alex N

    2015-09-17

    RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers. PMID:26320862

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

    PubMed

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

    2008-01-24

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

  2. Porcine circovirus type 2 replication is impaired by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway

    SciTech Connect

    Wei Li; Liu Jue

    2009-03-30

    Postweaning multisystemic wasting syndrome, which is primarily caused by porcine circovirus type 2 (PCV2), is an emerging and important swine disease. We have recently shown that PCV2 induces nuclear factor kappa B activation and its activation is required for active replication, but the other cellular factors involved in PCV2 replication are not well defined. The extracellular signal-regulated kinase (ERK) which served as an important component of cellular signal transduction pathways has been shown to regulate many viral infections. In this report, we show that PCV2 activates ERK1/2 in PCV2-infected PK15 cells dependent on viral replication. The PCV2-induced ERK1/2 leads to phosphorylation of the ternary complex factor Elk-1, which kinetically paralleled ERK1/2 activation. Inhibition of ERK activation with U0126, a specific MEK1/2 inhibitor, significantly reduced viral progeny release. Investigations into the mechanism of ERK1/2 regulation revealed that inhibition of ERK activation leads to decreased viral transcription and lower virus protein expression. These data indicate that the ERK signaling pathway is involved in PCV2 infection and beneficial to PCV2 replication in the cultured cells.

  3. Contribution of PIP-5 kinase I{alpha} to raft-based Fc{gamma}RIIA signaling

    SciTech Connect

    Szymanska, Ewelina; Korzeniowski, Marek; Raynal, Patrick; Sobota, Andrzej; Kwiatkowska, Katarzyna

    2009-04-01

    Receptor Fc{gamma}IIA (Fc{gamma}RIIA) associates with plasma membrane rafts upon activation to trigger signaling cascades leading to actin polymerization. We examined whether compartmentalization of PI(4,5)P{sub 2} and PI(4,5)P{sub 2}-synthesizing PIP5-kinase I{alpha} to rafts contributes to Fc{gamma}RIIA signaling. A fraction of PIP5-kinase I{alpha} was detected in raft-originating detergent-resistant membranes (DRM) isolated from U937 monocytes and other cells. The DRM of U937 monocytes contained also a major fraction of PI(4,5)P{sub 2}. PIP5-kinase I{alpha} bound PI(4,5)P{sub 2}, and depletion of the lipid displaced PIP5-kinase I{alpha} from the DRM. Activation of Fc{gamma}RIIA in BHK transfectants led to recruitment of the kinase to the plasma membrane and enrichment of DRM in PI(4,5)P{sub 2}. Immunofluorescence studies revealed that in resting cells the kinase was associated with the plasma membrane, cytoplasmic vesicles and the nucleus. After Fc{gamma}RIIA activation, PIP5-kinase I{alpha} and PI(4,5)P{sub 2} co-localized transiently with the activated receptor at distinct cellular locations. Immunoelectron microscopy studies revealed that PIP5-kinase I{alpha} and PI(4,5)P{sub 2} were present at the edges of electron-dense assemblies containing activated Fc{gamma}RIIA in their core. The data suggest that activation of Fc{gamma}RIIA leads to membrane rafts coalescing into signaling platforms containing PIP5-kinase I{alpha} and PI(4,5)P{sub 2}.

  4. A novel Lyn-protein kinase Cδ/ε-protein kinase D axis is activated in B cells by signalosome-independent alternate pathway BCR signaling.

    PubMed

    Guo, Benchang; Rothstein, Thomas L

    2013-06-01

    BCR signaling initiates multiple activities critical for B-cell function. Recently, we identified an alternate BCR signaling pathway, induced by IL-4, that is signalosome-independent, unlike the classical signalosome-dependent pathway, and that leads to activation of the MAP kinase, ERK. Here we questioned whether alternate pathway signaling extends to other key downstream events, especially protein kinase D (PKD) activation. We found that in murine spleen-derived B cells the IL-4-induced alternate pathway for BCR signaling results in PKD and PKD substrate phosphorylation, and that alternate pathway phosphorylation of HDAC5/7 and other key substrates requires PKD. Furthermore, we found that tyrosine phosphorylation of PKCδ/ε occurs as a result of alternate but not classical pathway signaling and is required for phosphorylation of PKD and PKD substrates. This result identifies PKCδ/ε tyrosine phosphorylation as a unique outcome of the alternate pathway. The alternate pathway is mediated by Lyn that is not required for classical pathway signaling and we found that Lyn associates directly with PKCδ/ε and is required for phosphorylation of PKCδ/ε and of PKD. These findings indicate that IL-4 influences B-cell activation by inducing a novel signaling pathway from BCR to Lyn to PKCδ/ε to PKD.

  5. Phosphatidylinositol 4-phosphate 5-kinase α activation critically contributes to CD28-dependent signaling responses.

    PubMed

    Muscolini, Michela; Camperio, Cristina; Capuano, Cristina; Caristi, Silvana; Piccolella, Enza; Galandrini, Ricciarda; Tuosto, Loretta

    2013-05-15

    CD28 is one of the most relevant costimulatory receptors that deliver both TCR-dependent and TCR-independent signals regulating a wide range of signaling pathways crucial for cytokine and chemokine gene expressions, T cell survival, and proliferation. Most of the CD28-dependent signaling functions are initiated by the recruitment and activation of class IA PI3Ks, which catalyze the conversion of phosphatidylinositol 4,5-biphosphate (PIP2) into phosphatidylinositol 3,4,5-triphosphate, thus generating the docking sites for key signaling proteins. Hence, PIP2 is a crucial substrate in driving the PI3K downstream signaling pathways, and PIP2 turnover may be an essential regulatory step to ensure the activation of PI3K following CD28 engagement. Despite some data evidence that CD28 augments TCR-induced turnover of PIP2, its direct role in regulating PIP2 metabolism has never been assessed. In this study, we show that CD28 regulates PIP2 turnover by recruiting and activating phosphatidylinositol 4-phosphate 5-kinases α (PIP5Kα) in human primary CD4(+) T lymphocytes. This event leads to the neosynthesis of PIP2 and to its consumption by CD28-activated PI3K. We also evidenced that PIP5Kα activation is required for both CD28 unique signals regulating IL-8 gene expression as well as for CD28/TCR-induced Ca(2+) mobilization, NF-AT nuclear translocation, and IL-2 gene transcription. Our findings elucidate a novel mechanism that involves PIP5Kα as a key modulator of CD28 costimulatory signals.

  6. Stimulation of extracellular signal-regulated kinases and proliferation in the human gastric cancer cells KATO-III by obestatin.

    PubMed

    Pazos, Yolanda; Alvarez, Carlos J P; Camiña, Jesus P; Casanueva, Felipe F

    2007-12-01

    Obestatin, the ghrelin-associated peptide, activates cell proliferation in the gastric cancer cell line KATO-III. The results showed that this peptide induced cell proliferation by mitogen-activated kinase kinase/extracellular signal-regulated kinases1/2 (ERK1/2) phosphorylation. A sequential analysis of the obestatin transmembrane signalling pathway indicated that the ERK1/2 activity is partially blocked after preincubation of the cells with pertussis toxin, as well as by wortmannin (an inhibitor of phosphoinositide 3-kinase (PI3K)), staurosporine (an inhibitor of protein kinase C (PKC)) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2, which inhibits the non receptor tyrosine kinase Src). Upon administration of obestatin, the intracellular levels of phospho-PKCepsilon- and theta-isoenzymes rise with similar time-courses, from which PKCepsilon appears to be the responsible for ERK1/2 response. Based on the experimental data, a signalling pathway involving the consecutive activation of G(i), PI3K, novel PKCepsilon and Src for ERK1/2 activation is proposed. These results point to a functionally active peptide that regulates proliferation of the gastric cancer cells KATO-III. PMID:18365868

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

    PubMed Central

    Cinnamon, Einat; Sawala, Annick; Tittiger, Claus; Paroush, Ze'ev

    2016-01-01

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

  8. 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.

  9. Metabotropic NMDA receptor signaling couples Src family kinases to pannexin-1 during excitotoxicity.

    PubMed

    Weilinger, Nicholas L; Lohman, Alexander W; Rakai, Brooke D; Ma, Evelyn M M; Bialecki, Jennifer; Maslieieva, Valentyna; Rilea, Travis; Bandet, Mischa V; Ikuta, Nathan T; Scott, Lucas; Colicos, Michael A; Teskey, G Campbell; Winship, Ian R; Thompson, Roger J

    2016-03-01

    Overactivation of neuronal N-methyl-D-aspartate receptors (NMDARs) causes excitotoxicity and is necessary for neuronal death. In the classical view, these ligand-gated Ca(2+)-permeable ionotropic receptors require co-agonists and membrane depolarization for activation. We report that NMDARs signal during ligand binding without activation of their ion conduction pore. Pharmacological pore block with MK-801, physiological pore block with Mg(2+) or a Ca(2+)-impermeable NMDAR variant prevented NMDAR currents, but did not block excitotoxic dendritic blebbing and secondary currents induced by exogenous NMDA. NMDARs, Src kinase and Panx1 form a signaling complex, and activation of Panx1 required phosphorylation at Y308. Disruption of this NMDAR-Src-Panx1 signaling complex in vitro or in vivo by administration of an interfering peptide either before or 2 h after ischemia or stroke was neuroprotective. Our observations provide insights into a new signaling modality of NMDARs that has broad-reaching implications for brain physiology and pathology.

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

    PubMed

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

    2016-08-01

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

  11. Diacylglycerol kinase-δ regulates AMPK signaling, lipid metabolism, and skeletal muscle energetics.

    PubMed

    Jiang, Lake Q; de Castro Barbosa, Thais; Massart, Julie; Deshmukh, Atul S; Löfgren, Lars; Duque-Guimaraes, Daniella E; Ozilgen, Arda; Osler, Megan E; Chibalin, Alexander V; Zierath, Juleen R

    2016-01-01

    Decrease of AMPK-related signal transduction and insufficient lipid oxidation contributes to the pathogenesis of obesity and type 2 diabetes. Previously, we identified that diacylglycerol kinase-δ (DGKδ), an enzyme involved in triglyceride biosynthesis, is reduced in skeletal muscle from type 2 diabetic patients. Here, we tested the hypothesis that DGKδ plays a role in maintaining appropriate AMPK action in skeletal muscle and energetic aspects of contraction. Voluntary running activity was reduced in DGKδ(+/-) mice, but glycogen content and mitochondrial markers were unaltered, suggesting that DGKδ deficiency affects skeletal muscle energetics but not mitochondrial protein abundance. We next determined the role of DGKδ in AMPK-related signal transduction and lipid metabolism in isolated skeletal muscle. AMPK activation and signaling were reduced in DGKδ(+/-) mice, concomitant with impaired lipid oxidation and elevated incorporation of free fatty acids into triglycerides. Strikingly, DGKδ deficiency impaired work performance, as evident by altered force production and relaxation dynamics in response to repeated contractions. In conclusion, DGKδ deficiency impairs AMPK signaling and lipid metabolism, thereby highlighting the deleterious role of excessive lipid metabolites in the development of peripheral insulin resistance and type 2 diabetes pathogenesis. DGKδ deficiency also influences skeletal muscle energetics, which may lead to low physical activity levels in type 2 diabetes.

  12. Signal transduction in macrophages by glycosylphosphatidylinositols of Plasmodium, Trypanosoma, and Leishmania: Activation of protein tyrosine kinases and protein kinase C by inositolglycan and diacylglycerol moieties

    PubMed Central

    Tachado, Souvenir D.; Gerold, Peter; Schwarz, Ralph; Novakovic, Suzanna; McConville, Malcolm; Schofield, Louis

    1997-01-01

    The perturbation of various glycosylphosphatidylinositol (GPI)-anchored surface proteins imparts profound regulatory signals to macrophages, lymphocytes and other cell types. The specific contribution of the GPI moieties to these events however is unclear. This study demonstrates that purified GPIs of Plasmodium falciparum, Trypanosoma brucei, and Leishmania mexicana origin are sufficient to initiate signal transduction when added alone to host cells as chemically defined agonists. GPIs (10 nM–1 μM) induce rapid activation of the protein tyrosine kinase (PTK) p59hck in macrophages. The minimal structural requirement for PTK activation is the evolutionarily conserved core glycan sequence Manα1-2Manα1-6Manα1-4GlcN1-6myo-inositol. GPI-associated diacylglycerols independently activate the calcium-independent ɛ isoform of protein kinase C. Both signals collaborate in regulating the downstream NF-κB/rel-dependent gene expression of interleukin 1α, tumor necrosis factor (TNF) α, and inducible NO synthase. The alkylacylglycerol-containing iM4 GIPL of L. mexicana, however, is unable to activate protein kinase C and inhibits TNF expression in response to other agonists, establishing signaling specificity among structurally distinct GPIs. GPI alone appears sufficient to mimic the activities of malaria parasite extracts in the signaling pathway leading to TNF expression. A mAb to GPI blocks TNF induction by parasite extracts indicating that GPI is a necessary agent in this response. As protozoal GPIs are closely related to their mammalian counterparts, the data indicate that GPIs do indeed constitute a novel outside-in signaling system, acting as both agonists and second messenger substrates, and imparting at least two separate signals through the structurally distinct glycan and fatty acid domains. These activities may underlie aspects of pathology and immune regulation in protozoal infections. PMID:9108098

  13. Src Family Kinases Promote Silencing of ATR-Chk1 Signaling in Termination of DNA Damage Checkpoint*

    PubMed Central

    Fukumoto, Yasunori; Morii, Mariko; Miura, Takahito; Kubota, Sho; Ishibashi, Kenichi; Honda, Takuya; Okamoto, Aya; Yamaguchi, Noritaka; Iwama, Atsushi; Nakayama, Yuji; Yamaguchi, Naoto

    2014-01-01

    The DNA damage checkpoint arrests cell cycle progression to allow time for repair. Once DNA repair is completed, checkpoint signaling is terminated. Currently little is known about the mechanism by which checkpoint signaling is terminated, and the disappearance of DNA lesions is considered to induce the end of checkpoint signaling; however, here we show that the termination of checkpoint signaling is an active process promoted by Src family tyrosine kinases. Inhibition of Src activity delays recovery from the G2 phase DNA damage checkpoint following DNA repair. Src activity is required for the termination of checkpoint signaling, and inhibition of Src activity induces persistent activation of ataxia telangiectasia mutated (ATM)- and Rad3-related (ATR) and Chk1 kinases. Src-dependent nuclear protein tyrosine phosphorylation and v-Src expression suppress the ATR-mediated Chk1 and Rad17 phosphorylation induced by DNA double strand breaks or DNA replication stress. Thus, Src family kinases promote checkpoint recovery through termination of ATR- and Chk1-dependent G2 DNA damage checkpoint. These results suggest a model according to which Src family kinases send a termination signal between the completion of DNA repair and the initiation of checkpoint termination. PMID:24634213

  14. The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling.

    PubMed Central

    Chu, D H; Spits, H; Peyron, J F; Rowley, R B; Bolen, J B; Weiss, A

    1996-01-01

    The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs. However, there is a CD45-deficient cell line that can signal through its TCR. We have studied this cell line to identify a TCR signaling pathway that is independent of CD45 regulation. In the course of these experiments, we found that the Syk PTK, but not the ZAP-70 PTK, is able to mediate TCR signaling independently of CD45 and of Lck. For this function, Syk requires functional kinase and SH2 domains, as well as intact phosphorylation sites in the regulatory loop of its kinase domain. Thus, differential expression of Syk is likely to explain the paradoxical phenotypes of different CD45-deficient T cells. Finally, these results suggest differences in activation requirements between two closely related PTK family members, Syk and ZAP-70. The differential activities of these two kinases suggest that they may play distinct, rather than completely redundant, roles in lymphocyte signaling. Images PMID:8947048

  15. Hypoxia induces a phase transition within a kinase signaling network in cancer cells

    PubMed Central

    Wei, Wei; Shi, Qihui; Remacle, Francoise; Qin, Lidong; Shackelford, David B.; Shin, Young Shik; Mischel, Paul S.; Levine, R. D.; Heath, James R.

    2013-01-01

    Hypoxia is a near-universal feature of cancer, promoting glycolysis, cellular proliferation, and angiogenesis. The molecular mechanisms of hypoxic signaling have been intensively studied, but the impact of changes in oxygen partial pressure (pO2) on the state of signaling networks is less clear. In a glioblastoma multiforme (GBM) cancer cell model, we examined the response of signaling networks to targeted pathway inhibition between 21% and 1% pO2. We used a microchip technology that facilitates quantification of a panel of functional proteins from statistical numbers of single cells. We find that near 1.5% pO2, the signaling network associated with mammalian target of rapamycin (mTOR) complex 1 (mTORC1)—a critical component of hypoxic signaling and a compelling cancer drug target—is deregulated in a manner such that it will be unresponsive to mTOR kinase inhibitors near 1.5% pO2, but will respond at higher or lower pO2 values. These predictions were validated through experiments on bulk GBM cell line cultures and on neurosphere cultures of a human-origin GBM xenograft tumor. We attempt to understand this behavior through the use of a quantitative version of Le Chatelier’s principle, as well as through a steady-state kinetic model of protein interactions, both of which indicate that hypoxia can influence mTORC1 signaling as a switch. The Le Chatelier approach also indicates that this switch may be thought of as a type of phase transition. Our analysis indicates that certain biologically complex cell behaviors may be understood using fundamental, thermodynamics-motivated principles. PMID:23530221

  16. Hypoxia induces a phase transition within a kinase signaling network in cancer cells.

    PubMed

    Wei, Wei; Shi, Qihui; Remacle, Francoise; Qin, Lidong; Shackelford, David B; Shin, Young Shik; Mischel, Paul S; Levine, R D; Heath, James R

    2013-04-01

    Hypoxia is a near-universal feature of cancer, promoting glycolysis, cellular proliferation, and angiogenesis. The molecular mechanisms of hypoxic signaling have been intensively studied, but the impact of changes in oxygen partial pressure (pO2) on the state of signaling networks is less clear. In a glioblastoma multiforme (GBM) cancer cell model, we examined the response of signaling networks to targeted pathway inhibition between 21% and 1% pO2. We used a microchip technology that facilitates quantification of a panel of functional proteins from statistical numbers of single cells. We find that near 1.5% pO2, the signaling network associated with mammalian target of rapamycin (mTOR) complex 1 (mTORC1)--a critical component of hypoxic signaling and a compelling cancer drug target--is deregulated in a manner such that it will be unresponsive to mTOR kinase inhibitors near 1.5% pO2, but will respond at higher or lower pO2 values. These predictions were validated through experiments on bulk GBM cell line cultures and on neurosphere cultures of a human-origin GBM xenograft tumor. We attempt to understand this behavior through the use of a quantitative version of Le Chatelier's principle, as well as through a steady-state kinetic model of protein interactions, both of which indicate that hypoxia can influence mTORC1 signaling as a switch. The Le Chatelier approach also indicates that this switch may be thought of as a type of phase transition. Our analysis indicates that certain biologically complex cell behaviors may be understood using fundamental, thermodynamics-motivated principles. PMID:23530221

  17. Protein Kinase A-Dependent and -Independent Signaling Pathways Contribute to Cyclic AMP-Stimulated Proliferation

    PubMed Central

    Cass, Lisa A.; Summers, Scott A.; Prendergast, Gregory V.; Backer, Jonathan M.; Birnbaum, Morris J.; Meinkoth, Judy L.

    1999-01-01

    The effects of cyclic AMP (cAMP) on cell proliferation are cell type specific. Although the growth-inhibitory effects of cAMP have been well studied, much less is known regarding how cAMP stimulates proliferation. We report that cAMP stimulates proliferation through both protein kinase A (PKA)-dependent and PKA-independent signaling pathways and that phosphatidylinositol 3-kinase (PI3K) is required for cAMP-stimulated mitogenesis. In cells where cAMP is a mitogen, cAMP-elevating agents stimulate membrane ruffling, Akt phosphorylation, and p70 ribosomal S6 protein kinase (p70s6k) activity. cAMP effects on ruffle formation and Akt were PKA independent but sensitive to wortmannin. In contrast, cAMP-stimulated p70s6k activity was repressed by PKA inhibitors but not by wortmannin or microinjection of the N-terminal SH2 domain of the p85 regulatory subunit of PI3K, indicating that p70s6k and Akt can be regulated independently. Microinjection of highly specific inhibitors of PI3K or Rac1, or treatment with the p70s6k inhibitor rapamycin, impaired cAMP-stimulated DNA synthesis, demonstrating that PKA-dependent and -independent pathways contribute to cAMP-mediated mitogenesis. Direct elevation of PI3K activity through microinjection of an antibody that stimulates PI3K activity or stable expression of membrane-localized p110 was sufficient to confer hormone-independent DNA synthesis when accompanied by elevations in p70s6k activity. These findings indicate that multiple pathways contribute to cAMP-stimulated mitogenesis, only some of which are PKA dependent. Furthermore, they demonstrate that the ability of cAMP to stimulate both p70s6k- and PI3K-dependent pathways is an important facet of cAMP-regulated cell cycle progression. PMID:10454535

  18. Cadmium activates extracellular signal-regulated kinase 5 in HK-2 human renal proximal tubular cells

    SciTech Connect

    Kondo, Mio; Inamura, Hisako; Matsumura, Ken-ichi; Matsuoka, Masato

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Cadmium exposure induces ERK5 phosphorylation in HK-2 renal proximal tubular cells. Black-Right-Pointing-Pointer BIX02189 treatment suppresses cadmium-induced ERK5 but not ERK1/2 phosphorylation. Black-Right-Pointing-Pointer BIX02189 treatment suppresses cadmium-induced CREB and c-Fos phosphorylation. Black-Right-Pointing-Pointer ERK5 activation by cadmium exposure may play an anti-apoptotic role in HK-2 cells. -- Abstract: We examined the effects of cadmium chloride (CdCl{sub 2}) exposure on the phosphorylation and functionality of extracellular signal-regulated kinase 5 (ERK5), a recently identified member of the mitogen-activated protein kinase (MAPK) family, in HK-2 human renal proximal tubular cells. Following exposure to CdCl{sub 2}, ERK5 phosphorylation increased markedly, but the level of total ERK5 was unchanged. ERK5 phosphorylation following CdCl{sub 2} exposure was rapid and transient, similar to the time course of ERK1/2 phosphorylation. Treatment of HK-2 cells with the MAPK/ERK kinase 5 inhibitor, BIX02189, suppressed CdCl{sub 2}-induced ERK5 but not ERK1/2 phosphorylation. The CdCl{sub 2}-induced increase of phosphorylated cAMP response element-binding protein (CREB) and activating transcription factor-1 (ATF-1), as well as the accumulation of mobility-shifted c-Fos protein, were suppressed by BIX02189 treatment. Furthermore, BIX02189 treatment enhanced cleavage of poly(ADP-ribose) polymerase and increased the level of cytoplasmic nucleosomes in HK-2 cells exposed to CdCl{sub 2}. These findings suggest that ERK5 pathway activation by CdCl{sub 2} exposure might induce the phosphorylation of cell survival-transcription factors, such as CREB, ATF-1, and c-Fos, and may exert a partial anti-apoptotic role in HK-2 cells.

  19. Hormone-induced intercellular signal transfer dissociates cyclic AMP- dependent protein kinase

    PubMed Central

    1984-01-01

    We used co-cultures of porcine ovarian granulosa cells and mouse adrenocortical tumor cells (Y-1) to examine the kinetics of contact- dependent intercellular signal transfer and to assess the molecular mechanisms employed by this process. Exposure to follicle-stimulating hormone (FSH) caused cAMP-dependent protein kinase dissociation in granulosa cells and, with time, in Y-1 cells if, and only if, they contacted a responding granulosa cell. Y-1 cells close to a granulosa cell but not touching it failed to respond similarly. In reciprocal experiments, co-cultures were stimulated with adrenocorticotropic hormone (ACTH). Y-1 cells dissociated protein kinase as did granulosa cells in contact with Y-1 cells; however, granulosa cells that were not in contact with Y-1 cells failed to respond to the hormone. Fluorogenic steroids were secreted by Y-1 cells cultured alone and stimulated with ACTH, but were not secreted by cultures exposed to FSH. Neither hormone caused fluorogenic steroid production by granulosa cells. On the other hand these steroids were secreted in co-cultures stimulated with ACTH and to a lesser degree in co-cultures exposed to FSH. Autoradiography revealed that I125-FSH bound only to granulosa cells, never to Y-1 cells, even if they were in contact with an ovarian cell. The possibility of cell fusion was tested by experiments in which Y-1 cell membranes were labeled with cationized ferritin. These cells were then placed in co-culture with ovarian granulosa cells that had previously been allowed to ingest latex spheres. At regions of gap junctions between Y-1 and granulosa cells ferritin remained attached to the adrenal cell membrane and was never observed to migrate to the granulosa cell membrane. From these data, we conclude that hormone specific stimulation of one cell type leads to protein kinase dissociation in heterotypic partners only if they contact a hormone responsive cell. This signal transfer is bidirectional, exhibits temporal kinetics and

  20. Neuron-restrictive silencer factor (NRSF) represses cocaine- and amphetamine-regulated transcript (CART) transcription and antagonizes cAMP-response element-binding protein signaling through a dual NRSE mechanism.

    PubMed

    Zhang, Jing; Wang, Sihan; Yuan, Lin; Yang, Yinxiang; Zhang, Bowen; Liu, Qingbin; Chen, Lin; Yue, Wen; Li, Yanhua; Pei, Xuetao

    2012-12-14

    Cocaine- and amphetamine-regulated transcript (CART) peptide plays a pivotal role in neuroprotection against stroke-related brain injury. However, the regulatory mechanism on CART transcription, especially the repression mechanism, is not fully understood. Here, we show that the transcriptional repressor neuron-restrictive silencer elements (NRSF, also known as REST) represses CART expression through direct binding to two NRSF-binding elements (NRSEs) in the CART promoter and intron 1 (named pNRSE and iNRSE, respectively). EMSA show that NRSF binds to pNRSE and iNRSE directly in vitro. ChIP assays show that NRSF recruits differential co-repressor complexes including CoREST and HDAC1 to these NRSEs. The presence of both NRSEs is required for efficient repression of CART transcription as indicated by reporter gene assays. NRSF overexpression antagonizes forskolin-mediated up-regulation of CART mRNA and protein. Ischemia insult triggered by oxygen-glucose deprivation (OGD) enhances NRSF mRNA levels and then NRSF antagonizes the CREB signaling on CART activation, leading to augmented cell death. Depletion of NRSF in combination with forskolin treatment increases neuronal survival after ischemic insult. These findings reveal a novel dual NRSE mechanism by which NRSF represses CART expression and suggest that NRSF may serve as a therapeutic target for stroke treatment. PMID:23086924

  1. Neuron-restrictive Silencer Factor (NRSF) Represses Cocaine- and Amphetamine-regulated Transcript (CART) Transcription and Antagonizes cAMP-response Element-binding Protein Signaling through a Dual NRSE Mechanism*

    PubMed Central

    Zhang, Jing; Wang, Sihan; Yuan, Lin; Yang, Yinxiang; Zhang, Bowen; Liu, Qingbin; Chen, Lin; Yue, Wen; Li, Yanhua; Pei, Xuetao

    2012-01-01

    Cocaine- and amphetamine-regulated transcript (CART) peptide plays a pivotal role in neuroprotection against stroke-related brain injury. However, the regulatory mechanism on CART transcription, especially the repression mechanism, is not fully understood. Here, we show that the transcriptional repressor neuron-restrictive silencer elements (NRSF, also known as REST) represses CART expression through direct binding to two NRSF-binding elements (NRSEs) in the CART promoter and intron 1 (named pNRSE and iNRSE, respectively). EMSA show that NRSF binds to pNRSE and iNRSE directly in vitro. ChIP assays show that NRSF recruits differential co-repressor complexes including CoREST and HDAC1 to these NRSEs. The presence of both NRSEs is required for efficient repression of CART transcription as indicated by reporter gene assays. NRSF overexpression antagonizes forskolin-mediated up-regulation of CART mRNA and protein. Ischemia insult triggered by oxygen-glucose deprivation (OGD) enhances NRSF mRNA levels and then NRSF antagonizes the CREB signaling on CART activation, leading to augmented cell death. Depletion of NRSF in combination with forskolin treatment increases neuronal survival after ischemic insult. These findings reveal a novel dual NRSE mechanism by which NRSF represses CART expression and suggest that NRSF may serve as a therapeutic target for stroke treatment. PMID:23086924

  2. Agonism and Antagonism at the Insulin Receptor

    PubMed Central

    Knudsen, Louise; Hansen, Bo Falck; Jensen, Pia; Pedersen, Thomas Åskov; Vestergaard, Kirsten; Schäffer, Lauge; Blagoev, Blagoy; Oleksiewicz, Martin B.; Kiselyov, Vladislav V.; De Meyts, Pierre

    2012-01-01

    Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs). In the case of the IR, no natural ligand or insulin analogue has been shown to exhibit antagonistic properties, with the exception of a crosslinked insulin dimer (B29-B’29). However, synthetic monomeric or dimeric peptides targeting sites 1 or 2 of the IR were shown to be either agonists or antagonists. We found here that the S961 peptide, previously described to be an IR antagonist, exhibited partial agonistic effects in the 1–10 nM range, showing altogether a bell-shaped dose-response curve. Intriguingly, the agonistic effects of S961 were seen only on mitogenic endpoints (3H-thymidine incorporation), and not on metabolic endpoints (14C-glucose incorporation in adipocytes and muscle cells). The agonistic effects of S961 were observed in 3 independent cell lines, with complete concordance between mitogenicity (3H-thymidine incorporation) and phosphorylation of the IR and Akt. Together with the B29-B’29 crosslinked dimer, S961 is a rare example of a mixed agonist/antagonist for the human IR. A plausible mechanistic explanation based on the bivalent crosslinking model of IR activation is proposed. PMID:23300584

  3. Mitogen-activated protein kinases with distinct requirements for Ste5 scaffolding influence signaling specificity in Saccharomyces cerevisiae.

    PubMed

    Flatauer, Laura J; Zadeh, Sheena F; Bardwell, Lee

    2005-03-01

    Scaffold proteins are believed to enhance specificity in cell signaling when different pathways share common components. The prototype scaffold Ste5 binds to multiple components of the Saccharomyces cerevisiae mating pheromone response pathway, thereby conducting the mating signal to the Fus3 mitogen-activated protein kinase (MAPK). Some of the kinases that Ste5 binds to, however, are also shared with other pathways. Thus, it has been presumed that Ste5 prevents its bound kinases from transgressing into other pathways and protects them from intrusions from those pathways. Here we found that Fus3MAPK required Ste5 scaffolding to receive legitimate signals from the mating pathway as well as misdirected signals leaking from other pathways. Furthermore, increasing the cellular concentration of active Ste5 enhanced the channeling of inappropriate stimuli to Fus3. This aberrant signal crossover resulted in the erroneous induction of cell cycle arrest and mating. In contrast to Fus3, the Kss1 MAPK did not require Ste5 scaffolding to receive either authentic or leaking signals. Furthermore, the Ste11 kinase, once activated via Ste5, was able to signal to Kss1 independently of Ste5 scaffolding. These results argue that Ste5 does not act as a barrier that actively prevents signal crossover to Fus3 and that Ste5 may not effectively sequester its activated kinases away from other pathways. Rather, we suggest that specificity in this network is promoted by the selective activation of Ste5 and the distinct requirements of the MAPKs for Ste5 scaffolding. PMID:15713635

  4. Fasting potentiates the anticancer activity of tyrosine kinase inhibitors by strengthening MAPK signaling inhibition.

    PubMed

    Caffa, Irene; D'Agostino, Vito; Damonte, Patrizia; Soncini, Debora; Cea, Michele; Monacelli, Fiammetta; Odetti, Patrizio; Ballestrero, Alberto; Provenzani, Alessandro; Longo, Valter D; Nencioni, Alessio

    2015-05-20

    Tyrosine kinase inhibitors (TKIs) are now the mainstay of treatment in many types of cancer. However, their benefit is frequently short-lived, mandating the search for safe potentiation strategies. Cycles of fasting enhance the activity of chemo-radiotherapy in preclinical cancer models and dietary approaches based on fasting are currently explored in clinical trials. Whether combining fasting with TKIs is going to be potentially beneficial remains unknown. Here we report that starvation conditions increase the ability of commonly administered TKIs, including erlotinib, gefitinib, lapatinib, crizotinib and regorafenib, to block cancer cell growth, to inhibit the mitogen-activated protein kinase (MAPK) signaling pathway and to strengthen E2F-dependent transcription inhibition. In cancer xenografts models, both TKIs and cycles of fasting slowed tumor growth, but, when combined, these interventions were significantly more effective than either type of treatment alone. In conclusion, cycles of fasting or of specifically designed fasting-mimicking diets should be evaluated in clinical studies as a means to potentiate the activity of TKIs in clinical use.

  5. Regulation of cell signaling dynamics by the protein kinase-scaffold Ste5.

    PubMed

    Hao, Nan; Nayak, Sujata; Behar, Marcelo; Shanks, Ryan H; Nagiec, Michal J; Errede, Beverly; Hasty, Jeffrey; Elston, Timothy C; Dohlman, Henrik G

    2008-06-01

    Cell differentiation requires the ability to detect and respond appropriately to a variety of extracellular signals. Here we investigate a differentiation switch induced by changes in the concentration of a single stimulus. Yeast cells exposed to high doses of mating pheromone undergo cell division arrest. Cells at intermediate doses become elongated and divide in the direction of a pheromone gradient (chemotropic growth). Either of the pheromone-responsive MAP kinases, Fus3 and Kss1, promotes cell elongation, but only Fus3 promotes chemotropic growth. Whereas Kss1 is activated rapidly and with a graded dose-response profile, Fus3 is activated slowly and exhibits a steeper dose-response relationship (ultrasensitivity). Fus3 activity requires the scaffold protein Ste5; when binding to Ste5 is abrogated, Fus3 behaves like Kss1, and the cells no longer respond to a gradient or mate efficiently with distant partners. We propose that scaffold proteins serve to modulate the temporal and dose-response behavior of the MAP kinase. PMID:18538663

  6. The involvement of Gab1 and PI 3-kinase in {beta}1 integrin signaling in keratinocytes

    SciTech Connect

    Kuwano, Yoshihiro; Fujimoto, Manabu . E-mail: fujimoto-m@umin.ac.jp; Watanabe, Rei; Ishiura, Nobuko; Nakashima, Hiroko; Komine, Mayumi; Hamazaki, Tatsuo S.; Tamaki, Kunihiko; Okochi, Hitoshi

    2007-09-14

    The control of the stem cell compartment in epidermis is closely linked to the regulation of keratinocyte proliferation and differentiation. {beta}1 integrins are expressed 2-fold higher by stem cells than transit-amplifying cells. Signaling from these {beta}1 integrins is critical for the regulation of the epidermal stem cell compartment. To clarify the functional relevance of this differential expression of {beta}1 integrins, we established HaCaT cells with high {beta}1integrin expression by repeated flow cytometric sorting of this population from the parental cell line. In these obtained cells expressing {beta}1 integrins by 5-fold, MAPK activation was markedly increased. Regarding the upstream of MAPK, Gab1 phosphorylation was also higher with high {beta}1 integrin expression, while Shc phosphorylation was not altered. In addition, enhanced phosphatidylinositol 3-kinase activation was also observed. These observations suggest that Gab1 and phosphatidylinositol 3-kinase play pivotal roles in the {beta}1 integrin-mediated regulation of the epidermal stem cell compartment.

  7. Sphingosine kinase 1/sphingosine 1-phosphate signalling pathway as a potential therapeutic target of pulmonary hypertension

    PubMed Central

    Xing, Xi-Qian; Li, Yan-Li; Zhang, Yu-Xuan; Xiao, Yi; Li, Zhi-Dong; Liu, Li-Qiong; Zhou, Yu-Shan; Zhang, Hong-Yan; Liu, Yan-Hong; Zhang, Li-Hui; Zhuang, Min; Chen, Yan-Ping; Ouyang, Sheng-Rong; Wu, Xu-Wei; Yang, Jiao

    2015-01-01

    Pulmonary hypertension is characterized by extensive vascular remodelling, leading to increased pulmonary vascular resistance and eventual death due to right heart failure. The pathogenesis of pulmonary hypertension involves vascular endothelial dysfunction and disordered vascular smooth muscle cell (VSMC) proliferation and migration, but the exact processes remain unknown. Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid involved in a wide spectrum of biological processes. S1P has been shown to regulate VSMC proliferation and migration and vascular tension via a family of five S1P G-protein-coupled receptors (S1P1-SIP5). S1P has been shown to have both a vasoconstrictive and vasodilating effect. The S1P receptors S1P1 and S1P3 promote, while S1P2 inhibits VSMC proliferation and migration in vitro in response to S1P. Moreover, it has been reported recently that sphingosine kinase 1 and S1P2 inhibitors might be useful therapeutic agents in the treatment of empirical pulmonary hypertension. The sphingosine kinase 1/S1P signalling pathways may play a role in the pathogenesis of pulmonary hypertension. Modulation of this pathway may offer novel therapeutic strategies. PMID:26550106

  8. Sphingosine kinase 1/sphingosine 1-phosphate signalling pathway as a potential therapeutic target of pulmonary hypertension.

    PubMed

    Xing, Xi-Qian; Li, Yan-Li; Zhang, Yu-Xuan; Xiao, Yi; Li, Zhi-Dong; Liu, Li-Qiong; Zhou, Yu-Shan; Zhang, Hong-Yan; Liu, Yan-Hong; Zhang, Li-Hui; Zhuang, Min; Chen, Yan-Ping; Ouyang, Sheng-Rong; Wu, Xu-Wei; Yang, Jiao

    2015-01-01

    Pulmonary hypertension is characterized by extensive vascular remodelling, leading to increased pulmonary vascular resistance and eventual death due to right heart failure. The pathogenesis of pulmonary hypertension involves vascular endothelial dysfunction and disordered vascular smooth muscle cell (VSMC) proliferation and migration, but the exact processes remain unknown. Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid involved in a wide spectrum of biological processes. S1P has been shown to regulate VSMC proliferation and migration and vascular tension via a family of five S1P G-protein-coupled receptors (S1P1-SIP5). S1P has been shown to have both a vasoconstrictive and vasodilating effect. The S1P receptors S1P1 and S1P3 promote, while S1P2 inhibits VSMC proliferation and migration in vitro in response to S1P. Moreover, it has been reported recently that sphingosine kinase 1 and S1P2 inhibitors might be useful therapeutic agents in the treatment of empirical pulmonary hypertension. The sphingosine kinase 1/S1P signalling pathways may play a role in the pathogenesis of pulmonary hypertension. Modulation of this pathway may offer novel therapeutic strategies. PMID:26550106

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

    PubMed Central

    Kuo, Chen-Tzu; Chen, Bing-Chang; Yu, Chung-Chi; Weng, Chih-Ming; Hsu, Ming-Jen; Chen, Chien-Chih; Chen, Mei-Chieh; Teng, Che-Ming; Pan, Shiow-Lin; Bien, Mauo-Ying; Shih, Chung-Hung; Lin, Chien-Huang

    2009-01-01

    In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1) in denbinobin-induced apoptosis in human lung adenocarcinoma (A549) cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN), two antioxidants (N-acetyl-L-cysteine (NAC) and glutathione (GSH)), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS) production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis. PMID:19405983

  10. Antidepressants increase glial cell line-derived neurotrophic factor production through monoamine-independent activation of protein tyrosine kinase and extracellular signal-regulated kinase in glial cells.

    PubMed

    Hisaoka, Kazue; Takebayashi, Minoru; Tsuchioka, Mami; Maeda, Natsuko; Nakata, Yoshihiro; Yamawaki, Shigeto

    2007-04-01

    Recent studies show that neuronal and glial plasticity are important for therapeutic action of antidepressants. We previously reported that antidepressants increase glial cell line-derived neurotrophic factor (GDNF) production in rat C6 glioma cells (C6 cells). Here, we found that amitriptyline, a tricyclic antidepressant, increased both GDNF mRNA expression and release, which were selectively and completely inhibited by mitogen-activated protein kinase kinase inhibitors. Indeed, treatment of amitriptyline rapidly increased extracellular signal-regulated kinase (ERK) activity, as well as p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase activities. Furthermore, different classes of antidepressants also rapidly increased ERK activity. The extent of acute ERK activation and GDNF release were significantly correlated to each other in individual antidepressants, suggesting an important role of acute ERK activation in GDNF production. Furthermore, antidepressants increased the acute ERK activation and GDNF mRNA expression in normal human astrocytes as well as C6 cells. Although 5-hydroxytryptamine (serotonin) (5-HT), but not noradrenaline or dopamine, increased ERK activation and GDNF release via 5-HT2A receptors, ketanserin, a 5-HT2A receptor antagonist, did not have any effect on the amitriptyline-induced ERK activation. Thus, GDNF production by amitriptyline was independent of monoamine. Both of the amitriptyline-induced ERK activation and GDNF mRNA expression were blocked by genistein, a general protein tyrosine kinase (PTK) inhibitor. Actually, we found that amitriptyline acutely increased phosphorylation levels of several phosphotyrosine-containing proteins. Taken together, these findings indicate that ERK activation through PTK regulates antidepressant-induced GDNF production and that the GDNF production in glial cells may be a novel action of the antidepressant, which is independent of monoamine. PMID:17210798

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

    PubMed

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

    2013-09-20

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

  12. Diabetes Causes Bone Marrow Endothelial Barrier Dysfunction by Activation of the RhoA–Rho-Associated Kinase Signaling Pathway

    PubMed Central

    Mangialardi, Giuseppe; Katare, Rajesh; Oikawa, Atsuhiko; Meloni, Marco; Reni, Carlotta; Emanueli, Costanza; Madeddu, Paolo

    2013-01-01

    Objective Diabetes mellitus causes bone marrow (BM) microangiopathy. This study aimed to investigate the mechanisms responsible for BM endothelial dysfunction in diabetes mellitus. Methods and Results The analysis of differentially expressed transcripts in BM endothelial cells (BMECs) from type-1 diabetic and nondiabetic mice showed an effect of diabetes mellitus on signaling pathways controlling cell death, migration, and cytoskeletal rearrangement. Type-1 diabetic-BMECs displayed high reactive oxygen species levels, increased expression and activity of RhoA and its associated protein kinases Rho-associated kinase 1/Rho-associated kinase 2, and reduced Akt phosphorylation/activity. Likewise, diabetes mellitus impaired Akt-related BMEC functions, such as migration, network formation, and angiocrine factor-releasing activity, and increased vascular permeability. Moreover, high glucose disrupted BMEC contacts through Src tyrosine kinase phosphorylation of vascular endothelial cadherin. These alterations were prevented by constitutively active Akt (myristoylated Akt), Rho-associated kinase inhibitor Y-27632, and Src inhibitors. Insulin replacement restored BMEC abundance, as assessed by flow cytometry analysis of the endothelial marker MECA32, and endothelial barrier function in BM of type-1 diabetic mice. Conclusion Redox-dependent activation of RhoA/Rho-associated kinase and Src/vascular endothelial cadherin signaling pathways, together with Akt inactivation, contribute to endothelial dysfunction in diabetic BM. Metabolic control is crucial for maintenance of endothelial cell homeostasis and endothelial barrier function in BM of diabetic mice. PMID:23307872

  13. Protein Kinase A Signaling Is Inhibitory for Reprogramming into Pluripotent Stem Cells.

    PubMed

    Kim, Jong Soo; Hong, Yean Ju; Choi, Hyun Woo; Choi, Sol; Do, Jeong Tae

    2016-03-01

    Somatic cells may be reprogrammed into pluripotent cells by the ectopic expression of defined transcription factors. However, some of the hurdles that affect the generation of induced pluripotent stem cells include extremely low efficiency and slow reprogramming. In the present study, we examined the effects of small molecules on cellular reprogramming and found that 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), an analog of cyclic adenosine monophosphate (cAMP), improves the reprogramming efficiency of reprogrammable mouse fibroblasts induced with dox in serum replacement (SR) medium. Interestingly, treatment with 8-Br-cAMP in mouse embryonic stem cell culture conditions does not affect reprogramming into the pluripotent state; however, reprogramming efficiency is significantly enhanced by inhibition of protein kinase A (PKA) in SR medium. Therefore, our results suggest that PKA signaling is unnecessary and may in fact act as a barrier to reprogramming into pluripotent stem cells. PMID:26728702

  14. NADPH Oxidase Biology and the Regulation of Tyrosine Kinase Receptor Signaling and Cancer Drug Cytotoxicity

    PubMed Central

    Paletta-Silva, Rafael; Rocco-Machado, Nathália; Meyer-Fernandes, José Roberto

    2013-01-01

    The outdated idea that reactive oxygen species (ROS) are only dangerous products of cellular metabolism, causing toxic and mutagenic effects on cellular components, is being replaced by the view that ROS have several important functions in cell signaling. In aerobic organisms, ROS can be generated from different sources, including the mitochondrial electron transport chain, xanthine oxidase, myeloperoxidase, and lipoxygenase, but the only enzyme family that produces ROS as its main product is the NADPH oxidase family (NOX enzymes). These transfer electrons from NADPH (converting it to NADP−) to oxygen to make O2•−. Due to their stability, the products of NADPH oxidase, hydrogen peroxide, and superoxide are considered the most favorable ROS to act as signaling molecules. Transcription factors that regulate gene expression involved in carcinogenesis are modulated by NADPH oxidase, and it has emerged as a promising target for cancer therapies. The present review discusses the mechanisms by which NADPH oxidase regulates signal transduction pathways in view of tyrosine kinase receptors, which are pivotal to regulating the hallmarks of cancer, and how ROS mediate the cytotoxicity of several cancer drugs employed in clinical practice. PMID:23434665

  15. TLR4 Signaling augments monocyte chemotaxis by regulating G protein-coupled receptor kinase 2 translocation.

    PubMed

    Liu, Zheng; Jiang, Yong; Li, Yuehua; Wang, Juan; Fan, Liyan; Scott, Melanie J; Xiao, Guozhi; Li, Song; Billiar, Timothy R; Wilson, Mark A; Fan, Jie

    2013-07-15

    Monocytes are critical effector cells of the innate immune system that protect the host by migrating to inflammatory sites, differentiating to macrophages and dendritic cells, eliciting immune responses, and killing pathogenic microbes. MCP-1, also known as CCL2, plays an important role in monocyte activation and migration. The chemotactic function of MCP-1 is mediated by binding to the CCR2 receptor, a member of the G protein-coupled receptor (GPCR) family. Desensitization of GPCR chemokine receptors is an important regulator of the intensity and duration of chemokine stimulation. GPCR kinases (GRKs) induce GPCR phosphorylation, and this leads to GPCR desensitization. Regulation of subcellular localization of GRKs is considered an important early regulatory mechanism of GRK function and subsequent GPCR desensitization. Chemokines and LPS are both present during Gram-negative bacterial infection, and LPS often synergistically exaggerates leukocyte migration in response to chemokines. In this study, we investigated the role and mechanism of LPS-TLR4 signaling on the regulation of monocyte chemotaxis. We demonstrate that LPS augments MCP-1-induced monocyte migration. We also show that LPS, through p38 MAPK signaling, induces phosphorylation of GRK2 at serine 670, which, in turn, suppresses GRK2 translocation to the membrane, thereby preventing GRK2-initiated internalization and desensitization of CCR2 in response to MCP-1. This results in enhanced monocyte migration. These findings reveal a novel function for TLR4 signaling in promoting innate immune cell migration.

  16. Protein kinase C signalling during miracidium to mother sporocyst development in the helminth parasite, Schistosoma mansoni.

    PubMed

    Ludtmann, Marthe H R; Rollinson, David; Emery, Aidan M; Walker, Anthony J

    2009-09-01

    For schistosomes, development of the miracidium to mother sporocyst within a compatible molluscan host requires considerable physiological and morphological changes by the parasite. The molecular mechanisms controlling such development have not been explored extensively. To begin to elucidate the importance of kinase-mediated signal transduction to this process, the phosphorylation (activation) of protein kinase C (PKC) in larval stages of Schistosoma mansoni undergoing in vitro transformation was explored. Mining of the S. mansoni genomic database revealed two S. mansoni PKC proteins with high homology to human PKCbeta and containing the conserved autophosphorylation (activation) site represented by serine 660 of human PKCbeta(II). Western blotting with anti-phosphospecific antibodies directed to this site demonstrated that miracidia freshly-hatched from eggs possessed PKC (78kDa) which was phosphorylated (activated) when miracidia were exposed to phorbol ester, and dephosphorylated (inhibited) following exposure to the PKC inhibitor GF109203X. Miracidia treated with the phospholipase C (PLC) inhibitor U73122 also displayed decreased PKC phosphorylation. S. mansoni PKC was phosphorylated during the initial 24h development of miracidia into mother sporocysts; after 31h and 48h development, phosphorylation was reduced by 72% and 86%, respectively. Confocal microscopy of miracidia revealed phosphorylated PKC associated with the neural mass, excretory vesicle, tegument, ciliated plates, terebratorium and germinal cells; in larvae undergoing transformation for 31h, phosphorylated PKC was only occasionally detected, being present in regions likely corresponding to the ridge cyton. Inhibition of PKC in miracidia by GF109230X resulted in accelerated transformation, particularly to the postmiracidium stage; ciliated plates were also shed from developing larvae more rapidly. These results highlight the dynamic nature of PKC signalling during S. mansoni postembryonic

  17. Nucleolar localization signals of LIM kinase 2 function as a cell-penetrating peptide.

    PubMed

    Kobayashi, Nahoko; Niwa, Mikio; Hao, Yang; Yoshida, Tetsuhiko

    2010-12-01

    LIM Kinase 2 (LIMK2) is a LIM domain-containing protein kinase which regulates actin polymerization thorough phosphorylation of the actin depolymerizing factor cofilin. It is also known to function as a shuttle between the cytoplasm and nucleus in endothelial cells. A basic amino acid-rich motif in LIMK2 was previously identified to be responsible for this shuttling function, as a nucleolar localization signal (NoLS). Here it is shown that this nucleolar localization signal sequence also has the characteristic function of a cell-penetrating peptide (CPP). We synthesized LIMK2 NoLS-conjugated peptides and a protein and analyzed their cell-penetrating abilities in various types of cells. The BC-box motif of the Von Hippel-Lindau (VHL) protein was used for the peptide. This motif previously has been reported to be involved in the neural differentiation of bone marrow stromal cells and skin-derived precursor cells. Green fluorescence protein (GFP) was used as a large biologically active biomolecule for the protein. The LIMK2 NoLS-conjugated peptides and protein translocated across the cell membranes of fibroblast cells, neural stem cells, and even iPS cells. These results suggest that LIMK2 NoLS acts as a cell-penetrating peptide and its cell-penetrating ability is not restricted by cell type. Moreover, from an in vivo assay using a mouse brain, it was confirmed that NoLS has potential for transporting biomolecules across the blood-brain barrier. PMID:20937035

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

    PubMed

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

    2015-02-01

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

  19. Targeting Mitogen-Activated Protein Kinase Signaling in Mouse Models of Cardiomyopathy Caused by Lamin A/C Gene Mutations.

    PubMed

    Muchir, Antoine; Worman, Howard J

    2016-01-01

    The most frequently occurring mutations in the gene encoding nuclear lamin A and nuclear lamin C cause striated muscle diseases virtually always involving the heart. In this review, we describe the approaches and methods used to discover that cardiomyopathy-causing lamin A/C gene mutations increase MAP kinase signaling in the heart and that this plays a role in disease pathogenesis. We review different mouse models of cardiomyopathy caused by lamin A/C gene mutations and how transcriptomic analysis of one model identified increased cardiac activity of the ERK1/2, JNK, and p38α MAP kinases. We describe methods used to measure the activity of these MAP kinases in mouse hearts and then discuss preclinical treatment protocols using pharmacological inhibitors to demonstrate their role in pathogenesis. Several of these kinase inhibitors are in clinical development and could potentially be used to treat human subjects with cardiomyopathy caused by lamin A/C gene mutations.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  2. Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade.

    PubMed Central

    Benn, J; Schneider, R J

    1994-01-01

    Hepatitis B virus produces a small (154-amino acid) transcriptional transactivating protein, HBx, which is required for viral infection and has been implicated in virus-mediated liver oncogenesis. However, the molecular mechanism for HBx activity and its possible influence on cell proliferation have remained obscure. A number of studies suggest that HBx may stimulate transcription by indirectly activating transcription factors, possibly by influencing cell signaling pathways. We now present biochemical evidence that HBx activates Ras and rapidly induces a cytoplasmic signaling cascade linking Ras, Raf, and mitogen-activated protein kinase (MAP kinase), leading to transcriptional transactivation. HBx strongly elevates levels of GTP-bound Ras, activated and phosphorylated Raf, and tyrosine-phosphorylated and activated MAP kinase. Transactivation of transcription factor AP-1 by HBx is blocked by inhibition of Ras or Raf activities but not by inhibition of Ca(2+)- and diacylglycerol-dependent protein kinase C. HBx was also found to stimulate DNA synthesis in serum-starved cells. The hepatitis B virus HBx protein therefore stimulates Ras-GTP complex formation and promotes downstream signaling through Raf and MAP kinases, and may influence cell proliferation. Images PMID:7937954

  3. Regulation of Raf-1 kinase by TNF via its second messenger ceramide and cross-talk with mitogenic signalling.

    PubMed

    Müller, G; Storz, P; Bourteele, S; Döppler, H; Pfizenmaier, K; Mischak, H; Philipp, A; Kaiser, C; Kolch, W

    1998-02-01

    Raf-1 kinase is a central regulator of mitogenic signal pathways, whereas its general role in signal transduction of tumour necrosis factor (TNF) is less well defined. We have investigated mechanisms of Raf-1 regulation by TNF and its messenger ceramide in cell-free assays, insect and mammalian cell lines. In vitro, ceramide specifically bound to the purified catalytic domain and enhanced association with activated Ras proteins, but did not affect the kinase activity of Raf-1. Cell-permeable ceramides induced a marked increase of Ras-Raf-1 complexes in cells co-expressing Raf-1 and activated Ras. Likewise, a fast elevation of the endogeneous ceramide level, induced by TNF treatment of human Kym-1 rhabdomyosarcoma cells, was followed by stimulation of Ras-Raf-1 association without significant Raf-1 kinase activation. Failure of TNF or ceramide to induce Raf-1 kinase was observed in several TNF-responsive cell lines. Both TNF and exogeneous C6-ceramide interfered with the mitogenic activation of Raf-1 and ERK by epidermal growth factor and down-regulated v-Src-induced Raf-1 kinase activity. TNF also induced the translocation of Raf-1 from the cytosolic to the particulate fraction, indicating that this negative regulatory cross-talk occurs at the cell membrane. Interference with mitogenic signals at the level of Raf-1 could be an important initial step in TNF's cytostatic action.

  4. Signal pathways in up-regulation of chemokines by tyrosine kinase MER/NYK in prostate cancer cells.

    PubMed

    Wu, Yi-Mi; Robinson, Dan R; Kung, Hsing-Jien

    2004-10-15

    The AXL/UFO family of tyrosine kinases is characterized by a common N-CAM (neural adhesion molecule)-related extracellular domain and a common ligand, GAS6 (growth arrest-specific protein 6). Family members are prone to transcriptional regulation and carry out diverse functions including the regulation of cell adhesion, migration, phagocytosis, and survival. In this report, we describe a new role of MER/N-CAM-related kinase (NYK), a member of the AXL family of kinases, in the up-regulation of chemokines in prostate cancer cells. We show that NYK has elevated expression in a subset of tumor specimens and prostate cancer cell lines. Activation of NYK in the prostate cancer cell line DU145 does not cause a mitogenic effect; instead, it causes a differentiation phenotype. Microarray analysis revealed that NYK is a strong inducer of endocrine factors including interleukin (IL)-8 and several other angiogenic CXC chemokines as well as bone morphogenic factors. The dramatic increase of IL-8 expression is seen at both transcriptional and posttranscriptional levels. The downstream signals engaged by NYK were characterized, and those responsible for the up-regulation of IL-8 transcription were defined. In contrast to IL-1alpha, NYK-induced up-regulation of IL-8 in DU145 depends on the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase/Jun/Fos pathway, but not phosphoinositide 3'-kinase/nuclear factor-kappaB. These data define a new function of the AXL family of kinases and suggest a potential role of NYK in prostate cancer progression. PMID:15492251

  5. The PI 3-kinase and mTOR signaling pathways are important modulators of epithelial tubule formation.

    PubMed

    Walid, Shereaf; Eisen, Randi; Ratcliffe, Don R; Dai, Kezhi; Hussain, M Mahmood; Ojakian, George K

    2008-08-01

    Using MDCK cells as a model system, evidence is presented demonstrating that the signaling pathways mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI 3-kinase) play important roles in the regulation of epithelial tubule formation. Incubation of cells with collagen gel overlays induced early (4-8 h) reorganization of cells (epithelial remodeling) into three-dimensional multicellular tubular structures over 24 h. An MDCK cell line stably expressing the PH domain of Akt, a PI 3-kinase downstream effector, coupled to green fluorescent protein (GFP-Akt-PH) was used to determine the distribution of phosphatidyl inositol-3,4,5-P(3) (PIP(3)), a product of PI 3-kinase. GFP-Akt-PH was associated with lateral membranes in control cells. After incubation with collagen gel overlays, GFP-Akt-PH redistributed into the lamellipodia of migrating cells suggesting that PIP(3) plays a role in epithelial remodeling. Using the small molecule inhibitor LY-294002 that inhibits both mTOR and PI 3-kinase, we demonstrated that kinase activity was required for epithelial remodeling, disruption of cell junctions and subsequent modulation of tubule formation. Since the mTOR signaling pathway is downstream of PI 3-kinase, the effects of rapamycin, a specific mTOR inhibitor, on tubule formation were assessed. Rapamycin did not affect epithelial remodeling or GFP-Akt-PH redistribution but inhibited elongated tubule formation that occurred later (24 h) in morphogenesis. These results were further supported by using RNA interference to down-regulate mTOR and inhibit tubule formation. Our studies demonstrate that PI 3-kinase regulates early epithelial remodeling stages while mTOR modulates latter stages of tubule development. PMID:18366086

  6. A novel signaling pathway associated with Lyn, PI 3-kinase and Akt supports the proliferation of myeloma cells

    SciTech Connect

    Iqbal, Mohd S.; Tsuyama, Naohiro; Obata, Masanori; Ishikawa, Hideaki

    2010-02-12

    Interleukin-6 (IL-6) is a growth factor for human myeloma cells. We have recently found that in myeloma cells the activation of both signal transducer and activator of transcription (STAT) 3 and extracellular signal-regulated kinase (ERK) 1/2 is not sufficient for the IL-6-induced proliferation, which further requires the activation of the src family kinases, such as Lyn. Here we showed that the Lyn-overexpressed myeloma cell lines had the higher proliferative rate with IL-6 and the enhanced activation of the phosphatidylinositol (PI) 3-kinase and Akt. The IL-6-induced phosphorylation of STAT3 and ERK1/2 was not up-regulated in the Lyn-overexpressed cells, indicating that the Lyn-PI 3-kinase-Akt pathway is independent of these pathways. The PI 3-kinase was co-precipitated with Lyn in the Lyn-overexpressed cells of which proliferation with IL-6 was abrogated by the specific inhibitors for PI 3-kinase or Akt, suggesting that the activation of the PI 3-kinase-Akt pathway associated with Lyn is indeed related to the concomitant augmentation of myeloma cell growth. Furthermore, the decreased expression of p53 and p21{sup Cip1} proteins was observed in the Lyn-overexpressed cells, implicating a possible downstream target of Akt. This study identifies a novel IL-6-mediated signaling pathway that certainly plays a role in the proliferation of myeloma cells and this novel mechanism of MM tumor cell growth associated with Lyn would eventually contribute to the development of MM treatment.

  7. Hypoxia promotes drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling

    PubMed Central

    Zhao, Changfu; Zhang, Qiao; Yu, Tao; Sun, Shudong; Wang, Wenjun; Liu, Guangyao

    2016-01-01

    Purpose Drug resistance has been recognized to be a major obstacle to the chemotherapy for osteosarcoma. And the potential importance of hypoxia as a target to reverse drug resistance in osteosarcoma has been indicated, though the mechanism underlining such role is not clarified. The present study aims to investigate the role of hypoxia in the drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling. Experimental design We investigated the promotion of the resistance to doxorubicin of osteosarcoma MG-63 and U2-os cells in vitro, and then determined the role of hypoxia-inducible factor-1 (HIF-1)α and HIF-1β, the activation and regulatory role of AMPK in the osteosarcoma U2-os cells which were treated with doxorubicin under hypoxia. Results It was demonstrated that hypoxia significantly reduced the sensitivity of MG-63 and U2-os cells to doxorubicin, indicating an inhibited viability reduction and a reduced apoptosis promotion. And such reduced sensitivity was not associated with HIF-1α, though it was promoted by hypoxia in U2-os cells. Interestingly, the AMPK signaling was significantly promoted by hypoxia in the doxorubicin-treated U2-os cells, with a marked upregulation of phosphorylated AMPK (Thr 172) and phosphorylated acetyl-CoA carboxylase (ACC) (Ser 79), which were sensitive to the AMPK activator, AICAR and the AMPK inhibitor, Compound C. Moreover, the promoted AMPK activity by AICAR or the downregulated AMPK activity by Compound C significantly reduced or promoted the sensitivity of U2-os cells to doxorubicin. Conclusion The present study confirmed the AMPK signaling activation in the doxorubicin-treated osteosarcoma cells, in response to hypoxia, and the chemical upregulation or downregulation of AMPK signaling reduced or increased the chemo-sensitivity of osteosarcoma U2-os cells in vitro. Our study implies that AMPK inhibition might be a effective strategy to sensitize osteocarcoma cells to chemotherapy. PMID

  8. Role played by Disabled-2 in albumin induced MAP Kinase signalling

    SciTech Connect

    Diwakar, Ramaswamy Pearson, Alexander L.; Colville-Nash, Paul; Baines, Deborah L.; Dockrell, Mark E.C.

    2008-02-15

    Albumin has been shown to activate the mitogen activated protein kinase (MAPK) pathway in proximal tubular cells (PTECs) of the kidney. Megalin, the putative receptor for albumin has potential signalling properties. However, the mechanisms by which megalin signals are unclear. The adaptor phosphoprotein Disabled-2 (Dab2) is known to interact with the cytoplasmic tail of megalin and may be involved in albumin-mediated MAPK signalling. In this study, we investigated the role of Dab2 in albumin-mediated MAPK signalling and further studied the role of Dab2 in albumin-induced TGF{beta}-1 secretion, a MAPK dependent event. We used RNA interference to knockdown Dab2 protein abundance in HKC-8 cells a model of human PTECs. Albumin activated ERK1,2 and Elk-1 in a MEK-1 dependent manner and resulted in secretion of TGF{beta}-1. In the absence of albumin, knockdown of Dab2 resulted in a trend towards increase in pERK1,2 consistent with its putative role as an inhibitor of cell proliferation. However albumin-induced ERK1,2 activation was completely abolished by Dab2 knockdown. Dab2 knockdown did not however result in inhibition of albumin-induced TGF{beta}-1 secretion. These results suggest that Dab2 is a ligand dependent bi-directional regulator of ERK1,2 activity by demonstrating that in addition to its more traditional role as an inhibitor of ERK1,2 it may also activate ERK1,2.

  9. Elucidation of the Roles of the Src kinases in pancreatic acinar cell signaling

    PubMed Central

    Nuche-Berenguer, Bernardo; Moreno, Paola; Jensen, R. T.

    2014-01-01

    Recent studies report the Src-Family kinases(SFK’s) are important in a number of physiological and pathophysiological responses of pancreatic acinar cells(pancreatitis, growth, apoptosis), however, the role of SFKs in various signaling cascades important in mediating these cell functions is either not investigated or unclear. To address this we investigated the action of SFKs in these signaling cascades in rat pancreatic acini by modulating SFK activity using three methods:Adenovirus-induced expression of an inactive dominant-negative CSK(Dn-CSK-Advirus) or Wild-Type CSK(Wt-CSK-Advirus), which activate or inhibit SFK, respectively or using the chemical inhibitor, PP2, with its inactive control, PP3. CCK(0.3,100 nM) and TPA(1 µM) activated SFK and altered the activation of FAK proteins(PYK2, p125 FAK), adaptor proteins(p130CAS, paxillin), MAPK (p42/44, JNK, p38), Shc, PKC(PKD, MARCKS), Akt but not GSK3-β. Changes in SFK activity by using the three methods of altering SFK activity affected CCK/TPAs activation of SFK, PYK2, p125 FAK, p130CAS, Shc, paxillin, Akt but not p42/44, JNK, p38, PKC(PKD, MARCKS) or GSK3-β. With chemical inhibition the active SFK inhibitor, PP2, but not the inactive control analogue, PP3, showed these effects. For all stimulated changes pre-incubation with both adenoviruses showed similar effects to chemical inhibition of SFK activity. In conclusion, using three different approaches to altering Src activity allowed us to define fully for the first time the roles of SFKs in acinar cell signaling. Our results show that in pancreatic acinar cells, SFKs play a much wider role than previously reported in activating a number of important cellular signaling cascades shown to be important in mediating both acinar cell physiological and pathophysiological responses. PMID:25079913

  10. Peroxiredoxin 1 suppresses apoptosis via regulation of the apoptosis signal-regulating kinase 1 signaling pathway in human oral leukoplakia

    PubMed Central

    ZHANG, MIN; NIU, WENWEN; ZHANG, JIANFEI; GE, LIHUA; YANG, JING; SUN, ZHENG; TANG, XIAOFEI

    2015-01-01

    Peroxiredoxin 1 (Prx1) has a significant role in several malignant types of tumor. However, the role of Prx1 in oral leukoplakia (OLK) has remained to be elucidated. OLK is a common precancerous lesion of the oral mucosa that has a very high malignant transformation rate. The aim of the present study was to investigate the roles of Prx1, and its association with apoptosis signal-regulating kinase 1 (ASK1) and p38 in OLK. A total of 20 OLK samples and 10 normal oral mucosa samples were obtained from patients at the Beijing Stomatological Hospital (Beijing, China). The messenger RNA (mRNA) and protein expression levels of Prx1, ASK1 and p38 were determined by polymerase chain reaction and western blot analysis, respectively. Flow cytometry was used to detect cell apoptosis. The interaction between Prx1 and ASK1 was examined in H2O2-treated DOK cells by glutathione-S-transferase pull-down assays and by co-immunoprecipitation in vitro. Compared with those of the normal oral mucosa, the mRNA levels of Prx1, ASK1 and p38 were elevated in OLK tissues (P<0.05). The protein expression levels of Prx1, phosphorylated-ASK1 (p-ASK1) and p-p38 were also significantly enhanced in OLK tissues compared with those of the normal mucosa (P<0.05). In Prx1-knockdown DOK cells, ASK1 and p38 were activated, leading to enhanced levels of apoptosis in response to H2O2. No clear interaction between Prx1 and ASK1 was detected in H2O2-treated DOK cells. Prx1 was suggested to be involved in OLK pathogenesis by providing resistance against extracellular damages from oxidative stress via inhibition of the ASK1-induced apoptotic signaling pathway. Targeting Prx1 may provide a novel therapeutic strategy for the treatment of patients with OLK. PMID:26622762

  11. Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas[OPEN

    PubMed Central

    Evans, Bradley S.; Li, Jia; Liu, Yu; Diamond, Spencer

    2016-01-01

    The networks that govern carbon metabolism and control intracellular carbon partitioning in photosynthetic cells are poorly understood. Target of Rapamycin (TOR) kinase is a conserved growth regulator that integrates nutrient signals and modulates cell growth in eukaryotes, though the TOR signaling pathway in plants and algae has yet to be completely elucidated. We screened the unicellular green alga Chlamydomonas reinhardtii using insertional mutagenesis to find mutants that conferred hypersensitivity to the TOR inhibitor rapamycin. We characterized one mutant, vip1-1, that is predicted to encode a conserved inositol hexakisphosphate kinase from the VIP family that pyrophosphorylates phytic acid (InsP6) to produce the low abundance signaling molecules InsP7 and InsP8. Unexpectedly, the rapamycin hypersensitive growth arrest of vip1-1 cells was dependent on the presence of external acetate, which normally has a growth-stimulatory effect on Chlamydomonas. vip1-1 mutants also constitutively overaccumulated triacylglycerols (TAGs) in a manner that was synergistic with other TAG inducing stimuli such as starvation. vip1-1 cells had reduced InsP7 and InsP8, both of which are dynamically modulated in wild-type cells by TOR kinase activity and the presence of acetate. Our data uncover an interaction between the TOR kinase and inositol polyphosphate signaling systems that we propose governs carbon metabolism and intracellular pathways that lead to storage lipid accumulation. PMID:27600537

  12. Tyrosine Phosphorylation of the BRI1 Receptor Kinase Emerges as a Component of Brassinosteriod Signaling in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brassinosteroids (BRs) are essential growth-promoting hormones that regulate many aspects of plant growth and development. Two leucine-rich repeat receptor-like kinases (LRR-RLKs) are involved in BR perception and signal transduction: BRASSINOSTEROID INSENSITIVE 1 (BRI1), which is the BR receptor, ...

  13. Hydrogen peroxide induces activation of insulin signaling pathway via AMP-dependent kinase in podocytes

    SciTech Connect

    Piwkowska, Agnieszka; Rogacka, Dorota; Angielski, Stefan; Jankowski, Maciej

    2012-11-09

    Highlights: Black-Right-Pointing-Pointer H{sub 2}O{sub 2} activates the insulin signaling pathway and glucose uptake in podocytes. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} induces time-dependent changes in AMPK phosphorylation. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} enhances insulin signaling pathways via AMPK activation. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} stimulation of glucose uptake is AMPK-dependent. -- Abstract: Podocytes are cells that form the glomerular filtration barrier in the kidney. Insulin signaling in podocytes is critical for normal kidney function. Insulin signaling is regulated by oxidative stress and intracellular energy levels. We cultured rat podocytes to investigate the effects of hydrogen peroxide (H{sub 2}O{sub 2}) on the phosphorylation of proximal and distal elements of insulin signaling. We also investigated H{sub 2}O{sub 2}-induced intracellular changes in the distribution of protein kinase B (Akt). Western blots showed that H{sub 2}O{sub 2} (100 {mu}M) induced rapid, transient phosphorylation of the insulin receptor (IR), the IR substrate-1 (IRS1), and Akt with peak activities at 5 min ({Delta} 183%, P < 0.05), 3 min ({Delta} 414%, P < 0.05), and 10 min ({Delta} 35%, P < 0.05), respectively. Immunostaining cells with an Akt-specific antibody showed increased intensity at the plasma membrane after treatment with H{sub 2}O{sub 2}>. Furthermore, H{sub 2}O{sub 2} inhibited phosphorylation of the phosphatase and tensin homologue (PTEN; peak activity at 10 min; {Delta} -32%, P < 0.05) and stimulated phosphorylation of the AMP-dependent kinase alpha subunit (AMPK{alpha}; 78% at 3 min and 244% at 10 min). The stimulation of AMPK was abolished with an AMPK inhibitor, Compound C (100 {mu}M, 2 h). Moreover, Compound C significantly reduced the effect of H{sub 2}O{sub 2} on IR phosphorylation by about 40% (from 2.07 {+-} 0.28 to 1.28 {+-} 0.12, P < 0.05). In addition, H{sub 2}O{sub 2} increased glucose uptake in podocytes

  14. The cooked meat carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine activates the extracellular signal regulated kinase mitogen-activated protein kinase pathway.

    PubMed

    Creton, Stuart K; Zhu, Huijun; Gooderham, Nigel J

    2007-12-01

    During the cooking of meat, mutagenic and carcinogenic heterocyclic amines are formed, the most abundant of which, 2-amino-1-methyl-6-phenylimidazo[4-5-b]pyridine (PhIP), induces tumors of the prostate, colon, and mammary gland in rats. Humans consuming cooked meat are exposed to PhIP on a daily basis, yet few studies have assessed the effects of PhIP at dietary relevant concentrations. In addition to its genotoxic properties, recent studies have shown that PhIP can activate estrogen receptor-mediated signaling pathways at doses that are similar to those that may be present in the body following consumption of a cooked meat meal. In the present study, we examined whether such doses of PhIP can affect estrogen receptor-independent signal transduction via the mitogen-activated protein kinase (MAPK) extracellular signal-related kinase (ERK) pathway to influence proliferation and migration in the human mammary epithelial cell line MCF10A and the prostate cancer cell line PC-3. At doses shown to have a proliferative effect on MCF10A cells (10(-11)-10(-7) mol/L), PhIP induced a rapid, transient increase in phosphorylation of both MAPK/ERK kinase 1/2 and ERKs. Inhibition of this pathway significantly reduced the PhIP-induced proliferation of MCF10A cells and the migration of PC-3 cells. The data presented here show that levels of PhIP that approximate to human dietary exposure stimulate cellular signaling pathways and result in increased growth and migration, processes linked to the promotion and progression of neoplastic disease. These findings provide strong evidence that PhIP acts as a tumor initiator and promoter and that dietary exposure to this compound could contribute to carcinogenesis in humans.

  15. Calcium-independent activation of extracellular signal-regulated kinases 1 and 2 by cyclic strain

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

    We have previously demonstrated that cyclic strain induces extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation in endothelial cells (EC). The aim of this study was to investigate the effect of Ca2+ on the activation of ERK1/2. Bovine aortic EC were pretreated with a chelator of extracellular Ca2+, ethylaneglycol-bis(aminoethylether)-tetra-acetate (EGTA), a depleter of Ca2+ pools, 2,5-Di-(tert-butyl)-1,4-benzohydroquinone (BHQ), or a Ca2+ channel blocker, GdCl3, and subjected to an average 10 % strain at a rate of 60 cycles/min for 10 min. BHQ and GdCl3 did not inhibit the strain-induced ERK1/2 activation. Chelation of normal extracellular Ca2+ (1.8 mM) medium with EGTA (3 mM) acutely stimulated baseline phosphorylation and activation of ERK1/2, thereby obscuring any strain-induced activation of ERK1/2. However, in EC preincubated for 24 hours in Ca2+-free medium, elevated baseline phosphorylation was minimally activated by EGTA (200 microM) such that cyclic strain stimulated ERK1/2 in the presence or absence of BHQ. These results suggest a Ca2+ independence of the ERK1/2 signaling pathway by cyclic strain. Copyright 1998 Academic Press.

  16. Opposing actions of TGF{beta} and MAP kinase signaling in undifferentiated hen granulosa cells

    SciTech Connect

    Woods, Dori C.; Haugen, Morgan J.; Johnson, A.L. . E-mail: johnson.128@nd.edu

    2005-10-21

    The present studies were conducted to establish interactions between transforming growth factor (TGF)-{beta} and the epidermal growth factor (EGF) family members, TGF{alpha} and betacellulin (BTC), relative to proliferation and differentiation of granulosa cells in hen ovarian follicles. Results presented demonstrate expression of TGF{beta} isoforms, plus TGF{alpha}, BTC, and ErbB receptors in prehierarchal follicles, thus establishing the potential for autocrine/paracrine signaling and cross-talk within granulosa cells at the onset of differentiation. Treatment with TGF{alpha} or BTC increases levels of TGF{beta}1 mRNA in undifferentiated granulosa cells, while the selective inhibitor of mitogen activated protein kinase signaling, U0126, reverses these effects. Moreover, TGF{beta}1 attenuates c-myc mRNA expression and granulosa cell proliferation, while TGF{alpha} blocks both these inhibitory effects. Collectively, these data provide evidence that EGF family ligands regulate both the expression and biological actions of TGF{beta}1 in hen granulosa cells, and indicate that the timely interaction of these opposing factors is an important modulator of both granulosa cell proliferation and differentiation.

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

    PubMed Central

    Christianson, John P.; Flyer-Adams, Johanna G.; Drugan, Robert C.; Amat, Jose; Daut, Rachel A.; Foilb, Allison R.; Watkins, Linda R.; Maier, Steven F.

    2014-01-01

    Behaviorally controllable stressors confer protection from the neurochemical and behavioral consequences of future uncontrollable stressors, a phenomenon termed “behavioral immunization”. Recent data implicate protein synthesis within the ventromedial prefrontal cortex (mPFC) as critical to behavioral immunization. Adult, male Sprague-Dawley rats were exposed to a series of controllable tailshocks and 1 week later to uncontrollable tailshocks, followed 24 h later by social exploration and shuttlebox escape tests. To test the involvement of N-methyl-D-aspartate receptors (NMDARs) and the extracellular signal-regulated kinase (ERK) cascade in behavioral immunization, either D-AP5 or the MEK inhibitor U0126 was injected to the prelimbic (PL) or infralimbic (IL) mPFC prior to controllable stress exposure. Phosphorylated ERK and P70S6K, regulators of transcription and translation, were quantified by Western blot or immunohistochemistry after controllable or uncontrollable tailshocks. Prior controllable stress prevented the social exploration and shuttlebox performance deficits caused by the later uncontrollable stressor, and this effect was blocked by injections of D-AP5 into mPFC. A significant increase in phosphorylated ERK1 and ERK2, but not P70S6K, occurred within the PL and IL in rats exposed to controllable stress, but not to uncontrollable stress. However, U0126 only prevented behavioral immunization when injected to the PL. We provide evidence that NMDAR and ERK dependent signaling within the PL region is required for behavioral immunization, a learned form of stressor resistance. PMID:25324750

  18. Regulation of tooth number by fine-tuning levels of receptor-tyrosine kinase signaling.

    PubMed

    Charles, Cyril; Hovorakova, Maria; Ahn, Youngwook; Lyons, David B; Marangoni, Pauline; Churava, Svatava; Biehs, Brian; Jheon, Andrew; Lesot, Hervé; Balooch, Guive; Krumlauf, Robb; Viriot, Laurent; Peterkova, Renata; Klein, Ophir D

    2011-09-01

    Much of our knowledge about mammalian evolution comes from examination of dental fossils, because the highly calcified enamel that covers teeth causes them to be among the best-preserved organs. As mammals entered new ecological niches, many changes in tooth number occurred, presumably as adaptations to new diets. For example, in contrast to humans, who have two incisors in each dental quadrant, rodents only have one incisor per quadrant. The rodent incisor, because of its unusual morphogenesis and remarkable stem cell-based continuous growth, presents a quandary for evolutionary biologists, as its origin in the fossil record is difficult to trace, and the genetic regulation of incisor number remains a largely open question. Here, we studied a series of mice carrying mutations in sprouty genes, the protein products of which are antagonists of receptor-tyrosine kinase signaling. In sprouty loss-of-function mutants, splitting of gene expression domains and reduced apoptosis was associated with subdivision of the incisor primordium and a multiplication of its stem cell-containing regions. Interestingly, changes in sprouty gene dosage led to a graded change in incisor number, with progressive decreases in sprouty dosage leading to increasing numbers of teeth. Moreover, the independent development of two incisors in mutants with large decreases in sprouty dosage mimicked the likely condition of rodent ancestors. Together, our findings indicate that altering genetic dosage of an antagonist can recapitulate ancestral dental characters, and that tooth number can be progressively regulated by changing levels of activity of a single signal transduction pathway.

  19. Regulation of Lipid Signaling by Diacylglycerol Kinases during T Cell Development and Function

    PubMed Central

    Krishna, Sruti; Zhong, Xiao-Ping

    2013-01-01

    Diacylglycerol (DAG) and phosphatidic acid (PA) are bioactive lipids synthesized when the T cell receptor binds to a cognate peptide-MHC complex. DAG triggers signaling by recruiting Ras guanyl-releasing protein 1, PKCθ, and other effectors, whereas PA binds to effector molecules that include mechanistic target of rapamycin, Src homology region 2 domain-containing phosphatase 1, and Raf1. While DAG-mediated pathways have been shown to play vital roles in T cell development and function, the importance of PA-mediated signals remains less clear. The diacylglycerol kinase (DGK) family of enzymes phosphorylates DAG to produce PA, serving as a molecular switch that regulates the relative levels of these critical second messengers. Two DGK isoforms, α and ζ, are predominantly expressed in T lineage cells and play an important role in conventional αβ T cell development. In mature T cells, the activity of these DGK isoforms aids in the maintenance of self-tolerance by preventing T cell hyper-activation and promoting T cell anergy. In this review, we discuss the roles of DAG-mediated pathways, PA-effectors, and DGKs in T cell development and function. We also highlight recent work that has uncovered previously unappreciated roles for DGK activity, for instance in invariant NKT cell development, anti-tumor and anti-viral CD8 responses, and the directional secretion of soluble effectors. PMID:23847619

  20. Sequential Transphosphorylation of the BRI1/BAK1 Receptor Kinase Pair Regulates Early Events of the Brassinosteriod Signaling Pathway Promoting Plant Growth and Development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brassinosteroids (BRs) regulate multiple aspects of plant growth and development through a signal transduction pathway that is initiated by BR binding to the transmembrane receptor kinase BRI1. Activated BRI1 heterodimerizes with a second receptor kinase, BAK1, leading to enhanced signaling output. ...

  1. Extracellular signal-regulated kinase phosphorylation in forebrain neurones contributes to osmoregulatory mechanisms

    PubMed Central

    Dine, Julien; Ducourneau, Vincent R R; Fénelon, Valérie S; Fossat, Pascal; Amadio, Aurélie; Eder, Matthias; Israel, Jean-Marc; Oliet, Stéphane H R; Voisin, Daniel L

    2014-01-01

    Vasopressin secretion from the magnocellular neurosecretory cells (MNCs) is crucial for body fluid homeostasis. Osmotic regulation of MNC activity involves the concerted modulation of intrinsic mechanosensitive ion channels, taurine release from local astrocytes as well as excitatory inputs derived from osmosensitive forebrain regions. Extracellular signal-regulated protein kinases (ERK) are mitogen-activated protein kinases that transduce extracellular stimuli into intracellular post-translational and transcriptional responses, leading to changes in intrinsic neuronal properties and synaptic function. Here, we investigated whether ERK activation (i.e. phosphorylation) plays a role in the functioning of forebrain osmoregulatory networks. We found that within 10 min after intraperitoneal injections of hypertonic saline (3 m, 6 m) in rats, many phosphoERK-immunopositive neurones were observed in osmosensitive forebrain regions, including the MNC containing supraoptic nuclei. The intensity of ERK labelling was dose-dependent. Reciprocally, slow intragastric infusions of water that lower osmolality reduced basal ERK phosphorylation. In the supraoptic nucleus, ERK phosphorylation predominated in vasopressin neurones vs. oxytocin neurones and was absent from astrocytes. Western blot experiments confirmed that phosphoERK expression in the supraoptic nucleus was dose dependent. Intracerebroventricular administration of the ERK phosphorylation inhibitor U 0126 before a hyperosmotic challenge reduced the number of both phosphoERK-immunopositive neurones and Fos expressing neurones in osmosensitive forebrain regions. Blockade of ERK phosphorylation also reduced hypertonically induced depolarization and an increase in firing of the supraoptic MNCs recorded in vitro. It finally reduced hypertonically induced vasopressin release in the bloodstream. Altogether, these findings identify ERK phosphorylation as a new element contributing to the osmoregulatory mechanisms of

  2. Involvement of Extracellular Signal Regulated Kinases in Traumatic Brain Injury-Induced Depression in Rodents

    PubMed Central

    Kuo, Jinn-Rung; Cheng, Yi-Hsuan; Chen, Yi-Shion

    2013-01-01

    Abstract Traumatic brain injury (TBI) is the most common cause of death and acquired disability among children and young adults in the developed countries. In clinical studies, the incidence of depression is high after TBI, and the mechanisms behind TBI-induced depression remain unclear. In the present study, we subjected rats to a moderate fluid percussion into the closed cranial cavity to induce TBI. After 3 days of recovery, injured rats were given a forced swim test (FST) and novelty-suppressed feeding tests. We found that TBI rats exhibited increased duration of immobility and longer latency to begin chewing food in a new environment compared with sham-operated rats. Western blot analysis showed that TBI led to a decrease in the phosphorylated levels of extracellular signal regulated kinases (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK). Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), significantly reduced the duration of immobility when administered once per day for 14 days. Consistent with behavioral tests, fluoxetine treatment reversed TBI-induced decrease in p-ERK1/2 and p-p38 MAPK levels. Pre-treatment with a selective tryptophan hydroxylase inhibitor para-chlorophenylalanine (PCPA) blocked the antidepressant effect of fluoxetine. PCPA also prevented the effect of fluoxetine on ERK1/2 phosphorylation without affecting p38 MAPK phosphorylation. Pre-treatment with ERK inhibitor SL327 but not p38 MAPK inhibitor SB203580 prevented the antidepressant effect of fluoxetine. These results suggest that ERK1/2 plays a critical role in TBI-induced depression. PMID:23360216

  3. Src Family Tyrosine Kinase Signaling Regulates FilGAP through Association with RBM10.

    PubMed

    Yamada, Hazuki; Tsutsumi, Koji; Nakazawa, Yuki; Shibagaki, Yoshio; Hattori, Seisuke; Ohta, Yasutaka

    2016-01-01

    FilGAP is a Rac-specific GTPase-activating protein (GAP) that suppresses lamellae formation. In this study, we have identified RBM10 (RNA Binding Motif domain protein 10) as a FilGAP-interacting protein. Although RBM10 is mostly localized in the nuclei in human melanoma A7 cells, forced expression of Src family tyrosine kinase Fyn induced translocation of RBM10 from nucleus into cell peripheries where RBM10 and FilGAP are co-localized. The translocation of RBM10 from nucleus appears to require catalytic activity of Fyn since kinase-negative Fyn mutant failed to induce translocation of RBM10 in A7 cells. When human breast carcinoma MDA-MB-231 cells are spreading on collagen-coated coverslips, endogenous FilGAP and RBM10 were localized at the cell periphery with tyrosine-phosphorylated proteins. RBM10 appears to be responsible for targeting FilGAP at the cell periphery because depletion of RBM10 by siRNA abrogated peripheral localization of FilGAP during cell spreading. Association of RBM10 with FilGAP may stimulate RacGAP activity of FilGAP. First, forced expression of RBM10 suppressed FilGAP-mediated cell spreading on collagen. Conversely, depletion of endogenous RBM10 by siRNA abolished FilGAP-mediated suppression of cell spreading on collagen. Second, FilGAP suppressed formation of membrane ruffles induced by Fyn and instead produced spiky cell protrusions at the cell periphery. This protrusive structure was also induced by depletion of Rac, suggesting that the formation of protrusions may be due to suppression of Rac by FilGAP. We found that depletion of RBM10 markedly reduced the formation of protrusions in cells transfected with Fyn and FilGAP. Finally, depletion of RBM10 blocked FilGAP-mediated suppression of ruffle formation induced by EGF. Taken together, these results suggest that Src family tyrosine kinase signaling may regulate FilGAP through association with RBM10. PMID:26751795

  4. Src Family Tyrosine Kinase Signaling Regulates FilGAP through Association with RBM10

    PubMed Central

    Yamada, Hazuki; Tsutsumi, Koji; Nakazawa, Yuki; Shibagaki, Yoshio; Hattori, Seisuke; Ohta, Yasutaka

    2016-01-01

    FilGAP is a Rac-specific GTPase-activating protein (GAP) that suppresses lamellae formation. In this study, we have identified RBM10 (RNA Binding Motif domain protein 10) as a FilGAP-interacting protein. Although RBM10 is mostly localized in the nuclei in human melanoma A7 cells, forced expression of Src family tyrosine kinase Fyn induced translocation of RBM10 from nucleus into cell peripheries where RBM10 and FilGAP are co-localized. The translocation of RBM10 from nucleus appears to require catalytic activity of Fyn since kinase-negative Fyn mutant failed to induce translocation of RBM10 in A7 cells. When human breast carcinoma MDA-MB-231 cells are spreading on collagen-coated coverslips, endogenous FilGAP and RBM10 were localized at the cell periphery with tyrosine-phosphorylated proteins. RBM10 appears to be responsible for targeting FilGAP at the cell periphery because depletion of RBM10 by siRNA abrogated peripheral localization of FilGAP during cell spreading. Association of RBM10 with FilGAP may stimulate RacGAP activity of FilGAP. First, forced expression of RBM10 suppressed FilGAP-mediated cell spreading on collagen. Conversely, depletion of endogenous RBM10 by siRNA abolished FilGAP-mediated suppression of cell spreading on collagen. Second, FilGAP suppressed formation of membrane ruffles induced by Fyn and instead produced spiky cell protrusions at the cell periphery. This protrusive structure was also induced by depletion of Rac, suggesting that the formation of protrusions may be due to suppression of Rac by FilGAP. We found that depletion of RBM10 markedly reduced the formation of protrusions in cells transfected with Fyn and FilGAP. Finally, depletion of RBM10 blocked FilGAP-mediated suppression of ruffle formation induced by EGF. Taken together, these results suggest that Src family tyrosine kinase signaling may regulate FilGAP through association with RBM10. PMID:26751795

  5. LKB1 kinase-dependent and -independent defects disrupt polarity and adhesion signaling to drive collagen remodeling during invasion

    PubMed Central

    Konen, Jessica; Wilkinson, Scott; Lee, Byoungkoo; Fu, Haian; Zhou, Wei; Jiang, Yi; Marcus, Adam I.

    2016-01-01

    LKB1 is a serine/threonine kinase and a commonly mutated gene in lung adenocarcinoma. The majority of LKB1 mutations are truncations that disrupt its kinase activity and remove its C-terminal domain (CTD). Because LKB1 inactivation drives cancer metastasis in mice and leads to aberrant cell invasion in vitro, we sought to determine how compromised LKB1 function affects lung cancer cell polarity and invasion. Using three-dimensional models, we show that LKB1 kinase activity is essential for focal adhesion kinase–mediated cell adhesion and subsequent collagen remodeling but not cell polarity. Instead, cell polarity is overseen by the kinase-independent function of its CTD and more specifically its farnesylation. This occurs through a mesenchymal-amoeboid morphological switch that signals through the Rho-GTPase RhoA. These data suggest that a combination of kinase-dependent and -independent defects by LKB1 inactivation creates a uniquely invasive cell with aberrant polarity and adhesion signaling that drives invasion into the microenvironment. PMID:26864623

  6. Periostin Promotes Atrioventricular Mesenchyme Matrix Invasion and Remodeling Mediated by Integrin Signaling through Rho/PI 3-Kinase

    PubMed Central

    Butcher, Jonathan T.; Norris, Russell A.; Hoffman, Stanley; Mjaatvedt, Corey H.; Markwald, Roger R.

    2007-01-01

    Recent evidence suggests that extracellular matrix components may play a signaling role in embryonic valve development. We have previously identified the spatiotemporal expression patterns of periostin in developing valves, but its function during this process is largely unknown. To evaluate the functional role periostin plays during valvulogenesis, two separate three-dimensional culture assay systems, which model chick atrioventricular cushion development, were employed. These assays demonstrated that cushion mesenchymal cells adhered and spread on purified periostin in a dose responsive manner, similar to collagen I and fibronectin via αvβ3 and β1 integrin pairs. Periostin overexpression resulted in enhanced mesenchyme invasion through 3D collagen gels and increased matrix compaction. This invasion was dependent on αvβ3 more than β1 integrin signaling, and was mediated differentially by Rho kinase and PI 3-kinase. Both matrix invasion and compaction were associated with a colocalization of periostin and β1 integrin expression to migratory cell phenotype in both surface and deep cells. The Rho/PI 3-kinase pathway also differentially mediated matrix compaction. Both Rho and PI 3-kinase were involved in normal cushion mesenchyme matrix compaction, but only PI 3-kinase was required for the enhanced matrix compaction due to periostin. Taken together, these results highlight periostin as a mediator of matrix remodeling by cushion mesenchyme towards a mature valve structure. PMID:17070513

  7. BR-SIGNALING KINASE1 Physically Associates with FLAGELLIN SENSING2 and Regulates Plant Innate Immunity in Arabidopsis[W

    PubMed Central

    Shi, Hua; Shen, Qiujing; Qi, Yiping; Yan, Haojie; Nie, Haozhen; Chen, Yongfang; Zhao, Ting; Katagiri, Fumiaki; Tang, Dingzhong

    2013-01-01

    Pathogen-associated molecular pattern (PAMP)-trigged immunity (PTI) is the first defensive line of plant innate immunity and is mediated by pattern recognition receptors. Here, we show that a mutation in BR-SIGNALING KINASE1 (BSK1), a substrate of the brassinosteroid (BR) receptor BRASSINOSTEROID INSENSITIVE1, suppressed the powdery mildew resistance caused by a mutation in ENHANCED DISEASE RESISTANCE2, which negatively regulates powdery mildew resistance and programmed cell death, in Arabidopsis thaliana. A loss-of-function bsk1 mutant displayed enhanced susceptibility to virulent and avirulent pathogens, including Golovinomyces cichoracearum, Pseudomonas syringae, and Hyaloperonospora arabidopsidis. The bsk1 mutant also accumulated lower levels of salicylic acid upon infection with G. cichoracearum and P. syringae. BSK1 belongs to a receptor-like cytoplasmic kinase family and displays kinase activity in vitro; this kinase activity is required for its function. BSK1 physically associates with the PAMP receptor FLAGELLIN SENSING2 and is required for a subset of flg22-induced responses, including the reactive oxygen burst, but not for mitogen-activated protein kinase activation. Our data demonstrate that BSK1 is involved in positive regulation of PTI. Together with previous findings, our work indicates that BSK1 represents a key component directly involved in both BR signaling and plant immunity. PMID:23532072

  8. Oncogenic tyrosine kinase NPM/ALK induces activation of the rapamycin-sensitive mTOR signaling pathway.

    PubMed

    Marzec, M; Kasprzycka, M; Liu, X; El-Salem, M; Halasa, K; Raghunath, P N; Bucki, R; Wlodarski, P; Wasik, M A

    2007-08-16

    The mechanisms of cell transformation mediated by the nucleophosmin (NPM)/anaplastic lymphoma kinase (ALK) tyrosine kinase are only partially understood. Here, we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma display persistent activation of mammalian target of rapamycin (mTOR) as determined by phosphorylation of mTOR targets S6rp and 4E-binding protein 1 (4E-BP1). The mTOR activation is serum growth factor-independent but nutrient-dependent. It is also dependent on the expression and enzymatic activity of NPM/ALK as demonstrated by cell transfection with wild-type and functionally deficient NPM/ALK, small interfering RNA (siRNA)-mediated NPM/ALK depletion and kinase activity suppression using the inhibitor WHI-P154. The NPM/ALK-induced mTOR activation is transduced through the mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway and, to a much lesser degree, through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Accordingly, whereas the low-dose PI3K inhibitor wortmannin and Akt inhibitor III profoundly inhibited Akt phosphorylation, they had a very modest effect on S6rp and 4E-BP1 phosphorylation. In turn, MEK inhibitors U0126 and PD98059 and siRNA-mediated depletion of either ERK1 or ERK2 inhibited S6rp phosphorylation much more effectively. Finally, the mTOR inhibitor rapamycin markedly decreased proliferation and increased the apoptotic rate of ALK+TCL cells. These findings identify mTOR as a novel key target of NPM/ALK and suggest that mTOR inhibitors may prove effective in therapy of ALK-induced malignancies.

  9. Combined computational and experimental analysis reveals mitogen-activated protein kinase-mediated feedback phosphorylation as a mechanism for signaling specificity.

    PubMed

    Hao, Nan; Yildirim, Necmettin; Nagiec, Michal J; Parnell, Stephen C; Errede, Beverly; Dohlman, Henrik G; Elston, Timothy C

    2012-10-01

    Different environmental stimuli often use the same set of signaling proteins to achieve very different physiological outcomes. The mating and invasive growth pathways in yeast each employ a mitogen-activated protein (MAP) kinase cascade that includes Ste20, Ste11, and Ste7. Whereas proper mating requires Ste7 activation of the MAP kinase Fus3, invasive growth requires activation of the alternate MAP kinase Kss1. To determine how MAP kinase specificity is achieved, we used a series of mathematical models to quantitatively characterize pheromone-stimulated kinase activation. In accordance with the computational analysis, MAP kinase feedback phosphorylation of Ste7 results in diminished activation of Kss1, but not Fus3. These findings reveal how feedback phosphorylation of a common pathway component can limit the activity of a competing MAP kinase through feedback phosphorylation of a common activator, and thereby promote signal fidelity. PMID:22875986

  10. cAMP Signaling Prevents Podocyte Apoptosis via Activation of Protein Kinase A and Mitochondrial Fusion

    PubMed Central

    Xie, Kewei; Ni, Zhaohui; Yan, Yucheng; Wei, Kai; Chuang, Peter Y.; He, John Cijiang; Gu, Leyi

    2014-01-01

    Our previous in vitro studies suggested that cyclic AMP (cAMP) signaling prevents adriamycin (ADR) and puromycin aminonucleoside (PAN)-induced apoptosis in podocytes. As cAMP is an important second messenger and plays a key role in cell proliferation, differentiation and cytoskeleton formation via protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac) pathways, we sought to determine the role of PKA or Epac signaling in cAMP-mediated protection of podocytes. In the ADR nephrosis model, we found that forskolin, a selective activator of adenylate cyclase, attenuated albuminuria and improved the expression of podocyte marker WT-1. When podocytes were treated with pCPT-cAMP (a selective cAMP/PKA activator), PKA activation was increased in a time-dependent manner and prevented PAN-induced podocyte loss and caspase 3 activation, as well as a reduction in mitochondrial membrane potential. We found that PAN and ADR resulted in a decrease in Mfn1 expression and mitochondrial fission in podocytes. pCPT-cAMP restored Mfn1 expression in puromycin or ADR-treated podocytes and induced Drp1 phosphorylation, as well as mitochondrial fusion. Treating podocytes with arachidonic acid resulted in mitochondrial fission, podocyte loss and cleaved caspase 3 production. Arachidonic acid abolished the protective effects of pCPT-cAMP on PAN-treated podocytes. Mdivi, a mitochondrial division inhibitor, prevented PAN-induced cleaved caspase 3 production in podocytes. We conclude that activation of cAMP alleviated murine podocyte caused by ADR. PKA signaling resulted in mitochondrial fusion in podocytes, which at least partially mediated the effects of cAMP. PMID:24642777

  11. Role of RhoA/Rho kinase signaling pathway in microgroove induced stem cell myogenic differentiation.

    PubMed

    Li, Huaqiong; Wen, Feng; Wang, Xincai; Tan, Lay Poh

    2015-06-01

    In our previous report, the authors have demonstrated that direct laser machined microchannels would trigger upregulation of myogenic markers in human mesenchymal stem cells (hMSCs) through promotion of cell elongation. However, the molecular basis signaling pathways behind this observation remains unclear. In this work, three types of microchannels generated by femtosecond laser were utilized to investigate possible mechanisms behind the induction of hMSCs myogenesis by microchannels. The authors hypothesized that small G-proteins RhoA and Rac1 play a vital role on myogenesis of hMSCs through regulating cytoskeleton rearrangement, via cell tension signaling cascades. The RhoA and Rac1 activities were evaluated for cells cultured on the micropatterned substrates, using a flat unpatterned substrate as control. It was found that significant activation of RhoA GTPase was exhibited for cells cultured on narrow microchannels (20-20-20 and 30-30-20), while no obvious differences were obtained on wide ones (80-30-20). Meanwhile, no significant difference was found for Rac1 activities on all tested groups. To further deduce the role of RhoA signaling pathway in microchannel directed stem cell myogenesis, the effectors of Rho, Rho kinase (ROCK) was chosen to explore how cell shape regulate myogenesis of hMSCs cultured on laser micropatterned substrate. A pharmacological ROCK inhibitor, Y-27632, was used to treat the cells and the effect on RhoA activation was investigated. Our data on the role of RhoA/ROCK in regulating cell myogenic differentiation on lasered microchannels substrates may provide a mechanistic insight on hMSCs fate directed by substrate topography.

  12. Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling.

    PubMed

    Alagramam, Kumar N; Stepanyan, Ruben; Jamesdaniel, Samson; Chen, Daniel H-C; Davis, Rickie R

    2014-01-01

    Noise-induced hearing loss (NIHL) is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL) broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4-8-week-old CBA/CaJ mice were exposed to no noise (control) or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK) signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b , Cacna1g , and Pla2g6 , related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway.

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  14. RhoA/phosphatidylinositol 3-kinase/protein kinase B/mitogen-activated protein kinase signaling after growth arrest-specific protein 6/mer receptor tyrosine kinase engagement promotes epithelial cell growth and wound repair via upregulation of hepatocyte growth factor in macrophages.

    PubMed

    Lee, Ye-Ji; Park, Hyun-Jung; Woo, So-Youn; Park, Eun-Mi; Kang, Jihee Lee

    2014-09-01

    Growth arrest-specific protein 6 (Gas6)/Mer receptor tyrosine kinase (Mer) signaling modulates cytokine secretion and helps to regulate the immune response and apoptotic cell clearance. Signaling pathways that activate an epithelial growth program in macrophages are still poorly defined. We report that Gas6/Mer/RhoA signaling can induce the production of epithelial growth factor hepatic growth factor (HGF) in macrophages, which ultimately promotes epithelial cell proliferation and wound repair. The RhoA/protein kinase B (Akt)/mitogen-activated protein (MAP) kinases, including p38 MAP kinase, extracellular signal-regulated protein kinase, and Jun NH2-terminal kinase axis in RAW 264.7 cells, was identified as Gas6/Mer downstream signaling pathway for the upregulation of HGF mRNA and protein. Conditioned medium from RAW 264.7 cells that had been exposed to Gas6 or apoptotic cells enhanced epithelial cell proliferation of the epithelial cell line LA-4 and wound closure. Cotreatment with an HGF receptor-blocking antibody or c-Met antagonist downregulated this enhancement. Inhibition of Mer with small interfering RNA (siRNA) or the RhoA/Rho kinase pathway by RhoA siRNA or Rho kinase pharmacologic inhibitor suppressed Gas6-induced HGF mRNA and protein expression in macrophages and blocked epithelial cell proliferation and wound closure induced by the conditioned medium. Our data provide evidence that macrophages can be reprogrammed by Gas6 to promote epithelial proliferation and wound repair via HGF, which is induced by the Mer/RhoA/Akt/MAP kinase pathway. Thus, defects in Gas6/Mer/RhoA signaling in macrophages may delay tissue repair after injury to the alveolar epithelium.

  15. Key signalling nodes in mammary gland development and cancer. Mitogen-activated protein kinase signalling in experimental models of breast cancer progression and in mammary gland development.

    PubMed

    Whyte, Jacqueline; Bergin, Orla; Bianchi, Alessandro; McNally, Sara; Martin, Finian

    2009-01-01

    Seven classes of mitogen-activated protein kinase (MAPK) intracellular signalling cascades exist, four of which are implicated in breast disease and function in mammary epithelial cells. These are the extracellular regulated kinase (ERK)1/2 pathway, the ERK5 pathway, the p38 pathway and the c-Jun N-terminal kinase (JNK) pathway. In some forms of human breast cancer and in many experimental models of breast cancer progression, signalling through the ERK1/2 pathway, in particular, has been implicated as being important. We review the influence of ERK1/2 activity on the organised three-dimensional association of mammary epithelial cells, and in models of breast cancer cell invasion. We assess the importance of epidermal growth factor receptor family signalling through ERK1/2 in models of breast cancer progression and the influence of ERK1/2 on its substrate, the oestrogen receptor, in this context. In parallel, we consider the importance of these MAPK-centred signalling cascades during the cycle of mammary gland development. Although less extensively studied, we highlight the instances of signalling through the p38, JNK and ERK5 pathways involved in breast cancer progression and mammary gland development.

  16. Modified high-density lipoprotein modulates aldosterone release through scavenger receptors via extra cellular signal-regulated kinase and Janus kinase-dependent pathways.

    PubMed

    Saha, Sarama; Graessler, Juergen; Schwarz, Peter E H; Goettsch, Claudia; Bornstein, Stefan R; Kopprasch, Steffi

    2012-07-01

    Patients with type 2 diabetes (T2D) manifest significant abnormalities in lipoprotein structure and function. The deleterious impact of oxidative and glycoxidative modifications on HDL-mediated atheroprotective, antiinflammatory, and antioxidative phenomena has been well established. However, the biological effects of modified HDL on adrenal steroidogenesis-which could reveal a pathophysiological link to the overactivity of the renin-angiotensin-aldosterone system and its adverse cardiovascular consequences often observed in T2D-are not well delineated. We studied the role of modified HDL on aldosterone release from adrenocortical carcinoma cells (NCI-H295R). In vitro modifications of native HDL were performed in the presence of glucose for glycoxidized HDL (glycoxHDL) and sodium hypochlorite for oxidized HDL. Angiotensin II (AngII)-sensitized H295R cells were treated with lipoproteins for 24 h, and supernatant was used to measure aldosterone release. Both native and modified HDL augmented the steroid release from AngII-sensitized cells, with glycoxHDL having the greatest impact. Both the modified forms of HDL induced a significant increase in scavenger receptor expression and employed protein kinase C as well as extracellular signal-regulated kinase as downstream effectors of aldosterone release. Native HDL and modified HDL required Janus kinase-2 for combating increased demand in steroidogenesis. Therefore, our data support the hypothesis that diabetes-induced modification of HDL may promote adrenocortical aldosterone secretion via different signal transduction pathways. This significant influence on multiple signaling mechanisms could be targeted for future research to implement novel therapeutic trials.

  17. Artocarpus altilis CG-901 alters critical nodes in the JH1-kinase domain of Janus kinase 2 affecting upstream JAK/STAT3 signaling.

    PubMed

    Nash, Oyekanmi; Omotuyi, Olaposi; Lee, Joonku; Kwon, Byoung-Mog; Ogbadu, Lucy

    2015-11-01

    As a key step in achieving low-cost, easily accessible anti-cancer therapy for low- and middle-income countries, we recently established the scientific basis for the folkloric use of Artocarpus altilis for the treatment of cancer by investigating the geranyl dihydrochalcone (CG-901) content and its interference with signal transducer and activator of transcription 3 (STAT3) phosphorylation and blockage of further downstream signaling. In the current study, the CG-901 upstream target was queried by chemical fingerprinting similarity assessment, semi-empirical (PM6ESCF) QMMM and molecular dynamics (MD) simulation. Moderate (∼0.4) to high (∼0.7) Tanimoto scores were found when the CG-901 scaffold was compared to ligands co-crystallized with Janus kinases (JAK) 1-3. High negative energy values were obtained when the CG-901 was treated semi-empirically (PM6ESCF) within the classical field of JAK (1-3). Multiple nanosecond MD simulations showed that CG-901 did not cause any large structural perturbations in the nucleotide-binding, activation and catalytic loops within the kinase (JH1) domain of JAK (1-3); however, it reduced the energy required to attain metastability along the path to energy minima conformation. In comparison to JAK1 and Apo-state JAK2, JAK2-bound CG-901 exhibited a highly re-organized key intra-domain protein network; indicating atomic level interference with inter-residue communication. In conclusion, CG-901 isolated from A. altilis represents a broad-spectrum JAK inhibitor, which may underlie the mechanism of STAT3 phosphorylation blockage. Graphical abstract Upper panel Janus kinase 2 upstream signaling pathway. Lower panel Apo-JAK2 (left) and CG-901-bound JAK2 (right).

  18. Artocarpus altilis CG-901 alters critical nodes in the JH1-kinase domain of Janus kinase 2 affecting upstream JAK/STAT3 signaling.

    PubMed

    Nash, Oyekanmi; Omotuyi, Olaposi; Lee, Joonku; Kwon, Byoung-Mog; Ogbadu, Lucy

    2015-11-01

    As a key step in achieving low-cost, easily accessible anti-cancer therapy for low- and middle-income countries, we recently established the scientific basis for the folkloric use of Artocarpus altilis for the treatment of cancer by investigating the geranyl dihydrochalcone (CG-901) content and its interference with signal transducer and activator of transcription 3 (STAT3) phosphorylation and blockage of further downstream signaling. In the current study, the CG-901 upstream target was queried by chemical fingerprinting similarity assessment, semi-empirical (PM6ESCF) QMMM and molecular dynamics (MD) simulation. Moderate (∼0.4) to high (∼0.7) Tanimoto scores were found when the CG-901 scaffold was compared to ligands co-crystallized with Janus kinases (JAK) 1-3. High negative energy values were obtained when the CG-901 was treated semi-empirically (PM6ESCF) within the classical field of JAK (1-3). Multiple nanosecond MD simulations showed that CG-901 did not cause any large structural perturbations in the nucleotide-binding, activation and catalytic loops within the kinase (JH1) domain of JAK (1-3); however, it reduced the energy required to attain metastability along the path to energy minima conformation. In comparison to JAK1 and Apo-state JAK2, JAK2-bound CG-901 exhibited a highly re-organized key intra-domain protein network; indicating atomic level interference with inter-residue communication. In conclusion, CG-901 isolated from A. altilis represents a broad-spectrum JAK inhibitor, which may underlie the mechanism of STAT3 phosphorylation blockage. Graphical abstract Upper panel Janus kinase 2 upstream signaling pathway. Lower panel Apo-JAK2 (left) and CG-901-bound JAK2 (right). PMID:26442513

  19. WRM-1 activates the LIT-1 protein kinase to transduce anterior/posterior polarity signals in C. elegans.

    PubMed

    Rocheleau, C E; Yasuda, J; Shin, T H; Lin, R; Sawa, H; Okano, H; Priess, J R; Davis, R J; Mello, C C

    1999-06-11

    During C. elegans development, Wnt/WG signaling is required for differences in cell fate between sister cells born from anterior/posterior divisions. A beta-catenin-related gene, wrm-1, and the lit-1 gene are effectors of this signaling pathway and appear to downregulate the activity of POP-1, a TCF/LEF-related protein, in posterior daughter cells. We show here that lit-1 encodes a serine/threonine protein kinase homolog related to the Drosophila tissue polarity protein Nemo. We demonstrate that the WRM-1 protein binds to LIT-1 in vivo and that WRM-1 can activate the LIT-1 protein kinase when coexpressed in vertebrate tissue culture cells. This activation leads to phosphorylation of POP-1 and to apparent changes in its subcellular localization. Our findings provide evidence for novel regulatory avenues for an evolutionarily conserved Wnt/WG signaling pathway. PMID:10380924

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

    PubMed

    Andrusiak, Matthew G; Jin, Yishi

    2016-04-01

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

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

    PubMed

    Andrusiak, Matthew G; Jin, Yishi

    2016-04-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. Role of Phosphatidylinositol Phosphate Signaling in the Regulation of the Filamentous-Growth Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Adhikari, Hema

    2015-01-01

    Reversible phosphorylation of the phospholipid phosphatidylinositol (PI) is a key event in the determination of organelle identity and an underlying regulatory feature in many biological processes. Here, we investigated the role of PI signaling in the regulation of the mitogen-activated protein kinase (MAPK) pathway that controls filamentous growth in yeast. Lipid kinases that generate phosphatidylinositol 4-phosphate [PI(4)P] at the Golgi (Pik1p) or PI(4,5)P2 at the plasma membrane (PM) (Mss4p and Stt4p) were required for filamentous-growth MAPK pathway signaling. Introduction of a conditional allele of PIK1 (pik1-83) into the filamentous (Σ1278b) background reduced MAPK activity and caused defects in invasive growth and biofilm/mat formation. MAPK regulatory proteins that function at the PM, including Msb2p, Sho1p, and Cdc42p, were mislocalized in the pik1-83 mutant, which may account for the signaling defects of the PI(4)P kinase mutants. Other PI kinases (Fab1p and Vps34p), and combinations of PIP (synaptojanin-type) phosphatases, also influenced the filamentous-growth MAPK pathway. Loss of these proteins caused defects in cell polarity, which may underlie the MAPK signaling defect seen in these mutants. In line with this possibility, disruption of the actin cytoskeleton by latrunculin A (LatA) dampened the filamentous-growth pathway. Various PIP signaling mutants were also defective for axial budding in haploid cells, cell wall construction, or proper regulation of the high-osmolarity glycerol response (HOG) pathway. Altogether, the study extends the roles of PI signaling to a differentiation MAPK pathway and other cellular processes. PMID:25724886

  4. Prolactin-Stimulated Activation of ERK1/2 Mitogen-Activated Protein Kinases is Controlled by PI3-Kinase/Rac/PAK Signaling Pathway in Breast Cancer Cells

    PubMed Central

    Aksamitiene, Edita; Achanta, Sirisha; Kolch, Walter; Kholodenko, Boris N.; Hoek, Jan B.; Kiyatkin, Anatoly

    2011-01-01

    There is strong evidence that deregulation of prolactin (PRL) signaling contributes to pathogenesis and chemoresistance of breast cancer. Therefore, understanding cross-talk between distinct signal transduction pathways triggered by activation of the prolactin receptor (PRL-R), is essential for elucidating the pathogenesis of metastatic breast cancer. In this study, we applied a sequential inhibitory analysis of various signaling intermediates to examine the hierarchy of protein interactions within the PRL signaling network and to evaluate the relative contributions of multiple signaling branches downstream of PRL-R to the activation of the extracellular signal-regulated kinases ERK1 and ERK2 in T47D and MCF-7 human breast cancer cells. Quantitative measurements of the phosphorylation/activation patterns of proteins showed that PRL simultaneously activated Src family kinases (SFKs) and the JAK/STAT, phosphoinositide-3 (PI3)-kinase/Akt and MAPK signaling pathways. The specific blockade or siRNA-mediated suppression of SFK/FAK, JAK2/STAT5, PI3-kinase/PDK1/Akt, Rac/PAK or Ras regulatory circuits revealed that (1) the PI3-kinase/Akt pathway is required for activation of the MAPK/ERK signaling cascade upon PRL stimulation; (2) PI3-kinase-mediated activation of the c-Raf-MEK1/2-ERK1/2 cascade occurs independent of signaling dowstream of STATs, Akt and PKC, but requires JAK2, SFKs and FAK activities; (3) activated PRL-R mainly utilizes the PI3-kinase-dependent Rac/PAK pathway rather than the canonical Shc/Grb2/SOS/Ras route to initiate and sustain ERK1/2 signaling. By interconnecting diverse signaling pathways PLR may enhance proliferation, survival, migration and invasiveness of breast cancer cells. PMID:21726627

  5. Muscarine enhances soluble amyloid precursor protein secretion in human neuroblastoma SH-SY5Y by a pathway dependent on protein kinase C(alpha), src-tyrosine kinase and extracellular signal-regulated kinase but not phospholipase C.

    PubMed

    Canet-Aviles, Rosa-Maria; Anderton, Mark; Hooper, Nigel M; Turner, Anthony J; Vaughan, Peter F T

    2002-06-15

    The signalling pathways by which muscarine and epidermal growth factor (EGF) regulate the secretion of the alpha-secretase cleavage product (sAPPalpha) of the amyloid precursor protein (APP) were examined in the human neuroblastoma SH-SY5Y. Using specific inhibitors it was found that over 80% of sAPPalpha secretion, enhanced by muscarine, occurred via the extracellular signal-regulated kinase (ERK1/2) member of the mitogen-activated protein kinase (MAPK) family and was dependent on protein kinase Calpha (PKCalpha) and a member of the Src family of non-receptor tyrosine kinases (Src-TK). In contrast the stimulation of sAPPalpha secretion by EGF was not affected by inhibitors of PKC nor Src-TK but was dependent on ERK1/2. In addition muscarine-enhanced sAPPalpha secretion and ERK1/2 activation were inhibited 60 and 80%, respectively, by micromolar concentrations of the phosphatidylinositol 3 kinase (PI-3K) inhibitor wortmannin. In comparison wortmannin decreased EGF stimulation of sAPPalpha secretion and ERK 1/2 activation by approximately 40%. Unexpectedly, U73122, an inhibitor of phosphoinositide-specific phospholipase C, did not inhibit muscarine enhancement of sAPPalpha secretion. These data are discussed in relation to a pathway for the enhancement of sAPPalpha secretion by muscarine which involves the activation of a Src-TK by G-protein beta/gamma-subunits leading to activation of PKCalpha, and ERK1/2 by a mechanism not involving phospholipase C. PMID:12191495

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

    PubMed

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

    2006-05-15

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

  7. A JAK2 interdomain linker relays Epo receptor engagement signals to kinase activation.

    PubMed

    Zhao, Lequn; Dong, Hongyun; Zhang, Cheng Cheng; Kinch, Lisa; Osawa, Mitsujiro; Iacovino, Michelina; Grishin, Nikolai V; Kyba, Michael; Huang, Lily Jun-shen

    2009-09-25

    JAK2 (Janus kinase 2) is essential for cytokine receptor signaling, and several lines of evidence support a causal role of an activating JAK2 mutation in myeloproliferative disorders. JAK2 activity is autoinhibited by its pseudokinase domain in the basal state, and the inhibition is released by cytokine stimulation; how engagement of the cognate receptor triggers this release is unknown. From a functional screen for gain-of-function JAK2 mutations, we discovered 13 missense mutations, nine in the pseudokinase domain and four in the Src homology 2 (SH2)-pseudokinase domain linker. These mutations identified determinants for autoinhibition and inducible activation in JAK2. Two of the mutants, K539I and N622I, resulted in erythrocytosis in mice. Scanning mutagenesis of the SH2-pseudokinase domain linker indicated that its N-terminal part was essential for interaction of JAK2 with the Epo receptor, whereas certain mutations in the C-terminal region conferred constitutive activation. We further showed that substitutions for Glu(543)-Asp(544) in this linker or Leu(611), Arg(683), or Phe(694) in the hinge proximal region of the pseudokinase domain resulted in activated JAK2 mutants that could not be further stimulated by Epo. These results suggest that the SH2-pseudokinase domain linker acts as a switch that relays cytokine engagement to JAK2 activation by flexing the pseudokinase domain hinge. PMID:19638629

  8. Context-dependent transcriptional interpretation of mitogen activated protein kinase signaling in the Drosophila embryo

    NASA Astrophysics Data System (ADS)

    Kim, Yoosik; Iagovitina, Antonina; Ishihara, Keisuke; Fitzgerald, Kate M.; Deplancke, Bart; Papatsenko, Dmitri; Shvartsman, Stanislav Y.

    2013-06-01

    Terminal regions of the Drosophila embryo are patterned by the localized activation of Mitogen Activated Protein Kinase (MAPK), which induces zygotic genes through relief of their repression by transcriptional repressor Capicua. The levels of MAPK activation at the anterior and posterior termini are close to each other, but the expression patterns of MAPK-target genes, such as zerknüllt (zen) and tailless (tll), display strong anterior-posterior (AP) asymmetry. This region-specific response to MAPK activation provides a clear example of context-dependent interpretation of inductive signaling, a common developmental effect that remains poorly understood. In the past, the AP asymmetry of zen expression was attributed to a mechanism that depends on MAPK substrate competition. We present data suggesting that the asymmetric expression of tll is generated by a different mechanism, based on feedforward control and multiple enhancers of the tll gene. A simple mathematical model of this mechanism correctly predicts how the wild-type expression pattern of tll changes in mutants affecting the anterior, dorsoventral, and terminal patterning systems and some of their direct targets.

  9. Trichoderma mitogen-activated protein kinase signaling is involved in induction of plant systemic resistance.

    PubMed

    Viterbo, Ada; Harel, Michal; Horwitz, Benjamin A; Chet, Ilan; Mukherjee, Prasun K

    2005-10-01

    The role of a mitogen-activated protein kinase (MAPK) TmkA in inducing systemic resistance in cucumber against the bacterial pathogen Pseudomonas syringae pv. lacrymans was investigated by using tmkA loss-of-function mutants of Trichoderma virens. In an assay where Trichoderma spores were germinated in proximity to cucumber roots, the mutants were able to colonize the plant roots as effectively as the wild-type strain but failed to induce full systemic resistance against the leaf pathogen. Interactions with the plant roots enhanced the level of tmkA transcript in T. virens and its homologue in Trichoderma asperellum. At the protein level, we could detect the activation of two forms reacting to the phospho-p44/42 MAPK antibody. Biocontrol experiments demonstrated that the tmkA mutants retain their biocontrol potential against Rhizoctonia solani in soil but are not effective against Sclerotium rolfsii in reducing disease incidence. Our results show that, unlike in many plant-pathogen interactions, Trichoderma TmkA MAPK is not involved in limited root colonization. Trichoderma, however, needs MAPK signaling in order to induce full systemic resistance in the plant.

  10. Intestinal Epithelial Cell Tyrosine Kinase 2 Transduces IL-22 Signals To Protect from Acute Colitis

    PubMed Central

    Hainzl, Eva; Rauch, Isabella; Heider, Susanne; Berry, David; Lassnig, Caroline; Schwab, Clarissa; Rosebrock, Felix; Milinovich, Gabriel; Schlederer, Michaela; Wagner, Michael; Schleper, Christa; Loy, Alexander; Urich, Tim; Kenner, Lukas; Han, Xiaonan; Decker, Thomas; Strobl, Birgit

    2015-01-01

    In the intestinal tract, IL-22 activates STAT3 to promote intestinal epithelial cell (IEC) homeostasis and tissue healing. The mechanism has remained obscure, but we demonstrate that IL-22 acts via tyrosine kinase 2 (Tyk2), a member of the Jak family. Using a mouse model for colitis, we show that Tyk2 deficiency is associated with an altered composition of the gut microbiota and exacerbates inflammatory bowel disease. Colitic Tyk2−/− mice have less p-STAT3 in colon tissue and their IECs proliferate less efficiently. Tyk2-deficient primary IECs show reduced p-STAT3 in response to IL-22 stimulation, and expression of IL-22–STAT3 target genes is reduced in IECs from healthy and colitic Tyk2−/− mice. Experiments with conditional Tyk2−/− mice reveal that IEC-specific depletion of Tyk2 aggravates colitis. Disease symptoms can be alleviated by administering high doses of rIL-22–Fc, indicating that Tyk2 deficiency can be rescued via the IL-22 receptor complex. The pivotal function of Tyk2 in IL-22–dependent colitis was confirmed in Citrobacter rodentium–induced disease. Thus, Tyk2 protects against acute colitis in part by amplifying inflammation-induced epithelial IL-22 signaling to STAT3. PMID:26432894

  11. Sex differences in fear extinction and involvements of extracellular signal-regulated kinase (ERK).

    PubMed

    Matsuda, Shingo; Matsuzawa, Daisuke; Ishii, Daisuke; Tomizawa, Haruna; Sutoh, Chihiro; Shimizu, Eiji

    2015-09-01

    Stress-related disorders, such as post-traumatic stress disorder (PTSD) and panic disorders, are disproportionately prevalent in females. However, the biological mechanism underlying these sex differences in the prevalence rate remains unclear. In the present study, we examined sex differences in fear memory, fear extinction, and spontaneous recovery of fear. We investigated the presence of sex differences in recent and remote fear memory in mice using contextual fear conditioning, as well as sex differences in spontaneous recovery of fear memory using a consecutive fear extinction paradigm. We examined the number of fear extinction days required to prevent spontaneous recovery of fear in either sex. We investigated whether ovariectomy affected fear extinction and spontaneous recovery. We also measured the activation of extracellular signal-regulated kinase (ERK) 1 and 2 in the dorsal hippocampus and the medial prefrontal cortex following fear extinction sessions. In our results, we found no sex difference in recent or remote fear memory. However, females required more fear extinction sessions compared to males to prevent spontaneous recovery. Within-extinction freezing also differed between males and females. Moreover, females required more extinction sessions than males to increase ERK2 phosphorylation in the dorsal hippocampus. Our data suggest that contextual fear extinction was unstable in females compared to males and that such sex differences may be related to the ERK2 phosphorylation in the hippocampus. PMID:26079214

  12. GSK621 Targets Glioma Cells via Activating AMP-Activated Protein Kinase Signalings

    PubMed Central

    Jiang, Hong; Liu, Wei; Zhan, Shi-Kun; Pan, Yi-Xin; Bian, Liu-Guan; Sun, Bomin; Sun, Qing-Fang; Pan, Si-Jian

    2016-01-01

    Here, we studied the anti-glioma cell activity by a novel AMP-activated protein kinase (AMPK) activator GSK621. We showed that GSK621 was cytotoxic to human glioma cells (U87MG and U251MG lines), possibly via provoking caspase-dependent apoptotic cell death. Its cytotoxicity was alleviated by caspase inhibitors. GSK621 activated AMPK to inhibit mammalian target of rapamycin (mTOR) and downregulate Tetraspanin 8 (Tspan8) in glioma cells. AMPK inhibition, through shRNA knockdown of AMPKα or introduction of a dominant negative (T172A) AMPKα, almost reversed GSK621-induced AMPK activation, mTOR inhibition and Tspan8 degradation. Consequently, GSK621’s cytotoxicity in glioma cells was also significantly attenuated by AMPKα knockdown or mutation. Further studies showed that GSK621, at a relatively low concentration, significantly potentiated temozolomide (TMZ)’s sensitivity and lethality against glioma cells. We summarized that GSK621 inhibits human glioma cells possibly via activating AMPK signaling. This novel AMPK activator could be a novel and promising anti-glioma cell agent. PMID:27532105

  13. Context-dependent transcriptional interpretation of mitogen activated protein kinase signaling in the Drosophila embryo

    PubMed Central

    Kim, Yoosik; Iagovitina, Antonina; Ishihara, Keisuke; Fitzgerald, Kate M.; Deplancke, Bart; Papatsenko, Dmitri; Shvartsman, Stanislav Y.

    2013-01-01

    Terminal regions of the Drosophila embryo are patterned by the localized activation of Mitogen Activated Protein Kinase (MAPK), which induces zygotic genes through relief of their repression by transcriptional repressor Capicua. The levels of MAPK activation at the anterior and posterior termini are close to each other, but the expression patterns of MAPK-target genes, such as zerknüllt (zen) and tailless (tll), display strong anterior-posterior (AP) asymmetry. This region-specific response to MAPK activation provides a clear example of context-dependent interpretation of inductive signaling, a common developmental effect that remains poorly understood. In the past, the AP asymmetry of zen expression was attributed to a mechanism that depends on MAPK substrate competition. We present data suggesting that the asymmetric expression of tll is generated by a different mechanism, based on feedforward control and multiple enhancers of the tll gene. A simple mathematical model of this mechanism correctly predicts how the wild-type expression pattern of tll changes in mutants affecting the anterior, dorsoventral, and terminal patterning systems and some of their direct targets. PMID:23822503

  14. Sex differences in fear extinction and involvements of extracellular signal-regulated kinase (ERK).

    PubMed

    Matsuda, Shingo; Matsuzawa, Daisuke; Ishii, Daisuke; Tomizawa, Haruna; Sutoh, Chihiro; Shimizu, Eiji

    2015-09-01

    Stress-related disorders, such as post-traumatic stress disorder (PTSD) and panic disorders, are disproportionately prevalent in females. However, the biological mechanism underlying these sex differences in the prevalence rate remains unclear. In the present study, we examined sex differences in fear memory, fear extinction, and spontaneous recovery of fear. We investigated the presence of sex differences in recent and remote fear memory in mice using contextual fear conditioning, as well as sex differences in spontaneous recovery of fear memory using a consecutive fear extinction paradigm. We examined the number of fear extinction days required to prevent spontaneous recovery of fear in either sex. We investigated whether ovariectomy affected fear extinction and spontaneous recovery. We also measured the activation of extracellular signal-regulated kinase (ERK) 1 and 2 in the dorsal hippocampus and the medial prefrontal cortex following fear extinction sessions. In our results, we found no sex difference in recent or remote fear memory. However, females required more fear extinction sessions compared to males to prevent spontaneous recovery. Within-extinction freezing also differed between males and females. Moreover, females required more extinction sessions than males to increase ERK2 phosphorylation in the dorsal hippocampus. Our data suggest that contextual fear extinction was unstable in females compared to males and that such sex differences may be related to the ERK2 phosphorylation in the hippocampus.

  15. Signaling between two interacting sensor kinases promotes biofilms and colonization by a bacterial symbiont

    PubMed Central

    Norsworthy, Allison N.; Visick, Karen L.

    2015-01-01

    Summary Cells acclimate to fluctuating environments by utilizing sensory circuits. One common sensory pathway used by bacteria is two-component signaling (TCS), composed of an environmental sensor (the sensor kinase, SK) and a cognate, intracellular effector (the response regulator, RR). The squid symbiont Vibrio fischeri uses an elaborate TCS phosphorelay containing a hybrid SK, RscS, and two RRs, SypE and SypG, to control biofilm formation and host colonization. Here, we found that another hybrid SK, SypF, was essential for biofilms by functioning downstream of RscS to directly control SypE and SypG. Surprisingly, although wild-type SypF functioned as a SK in vitro, this activity was dispensable for colonization. In fact, only a single non-enzymatic domain within SypF, the HPt domain, was critical in vivo. Remarkably, this domain within SypF interacted with RscS to permit a bypass of RscS’s own HPt domain and SypF’s enzymatic function. This represents the first in vivo example of a functional SK that exploits the enzymatic activity of another SK, an adaptation that demonstrates the elegant plasticity in the arrangement of TCS regulators. PMID:25586643

  16. Protein kinase Czeta regulates Cdk5/p25 signaling during myogenesis.

    PubMed

    de Thonel, Aurélie; Ferraris, Saima E; Pallari, Hanna-Mari; Imanishi, Susumu Y; Kochin, Vitaly; Hosokawa, Tomohisa; Hisanaga, Shin-ichi; Sahlgren, Cecilia; Eriksson, John E

    2010-04-15

    Atypical protein kinase Czeta (PKCzeta) is emerging as a mediator of differentiation. Here, we describe a novel role for PKCzeta in myogenic differentiation, demonstrating that PKCzeta activity is indispensable for differentiation of both C2C12 and mouse primary myoblasts. PKCzeta was found to be associated with and to regulate the Cdk5/p35 signaling complex, an essential factor for both neuronal and myogenic differentiation. Inhibition of PKCzeta activity prevented both myotube formation and simultaneous reorganization of the nestin intermediate filament cytoskeleton, which is known to be regulated by Cdk5 during myogenesis. p35, the Cdk5 activator, was shown to be a specific phosphorylation target of PKCzeta. PKCzeta-mediated phosphorylation of Ser-33 on p35 promoted calpain-mediated cleavage of p35 to its more active and stable fragment, p25. Strikingly, both calpain activation and the calpain-mediated cleavage of p35 were shown to be PKCzeta-dependent in differentiating myoblasts. Overall, our results identify PKCzeta as a controller of myogenic differentiation by its regulation of the phosphorylation-dependent and calpain-mediated p35 cleavage, which is crucial for the amplification of the Cdk5 activity that is required during differentiation.

  17. An allosteric signaling pathway of human 3-phosphoglycerate kinase from force distribution analysis.

    PubMed

    Palmai, Zoltan; Seifert, Christian; Gräter, Frauke; Balog, Erika

    2014-01-01

    3-Phosphogycerate kinase (PGK) is a two domain enzyme, which transfers a phosphate group between its two substrates, 1,3-bisphosphoglycerate bound to the N-domain and ADP bound to the C-domain. Indispensable for the phosphoryl transfer reaction is a large conformational change from an inactive open to an active closed conformation via a hinge motion that should bring substrates into close proximity. The allosteric pathway resulting in the active closed conformation has only been partially uncovered. Using Molecular Dynamics simulations combined with Force Distribution Analysis (FDA), we describe an allosteric pathway, which connects the substrate binding sites to the interdomain hinge region. Glu192 of alpha-helix 7 and Gly394 of loop L14 act as hinge points, at which these two secondary structure elements straighten, thereby moving the substrate-binding domains towards each other. The long-range allosteric pathway regulating hPGK catalytic activity, which is partially validated and can be further tested by mutagenesis, highlights the virtue of monitoring internal forces to reveal signal propagation, even if only minor conformational distortions, such as helix bending, initiate the large functional rearrangement of the macromolecule.

  18. Tyrosine phosphorylation is a mandatory proximal step in radiation-induced activation of the protein kinase C signaling pathway in human B-lymphocyte precursors.

    PubMed Central

    Uckun, F M; Schieven, G L; Tuel-Ahlgren, L M; Dibirdik, I; Myers, D E; Ledbetter, J A; Song, C W

    1993-01-01

    Ionizing radiation triggers a signal in human B-lymphocyte precursors that is intimately linked to an active protein-tyrosine kinase regulatory pathway. We show that in B-lymphocyte precursors, irradiation with gamma-rays leads to (i) stimulation of phosphatidylinositol turnover; (ii) downstream activation by covalent modification of multiple serine-specific protein kinases, including protein kinase C; and (iii) activation of nuclear factor kappa B. All of the radiation-induced signals were effectively prevented by the protein-tyrosine kinase inhibitors genistein and herbimycin A. Thus, tyrosine phosphorylation is an important and perhaps mandatory proximal step in the activation of the protein kinase C signaling cascade in human B-lymphocyte precursors. Our report expands current knowledge of the radiation-induced signaling cascade by clarifying the chronological sequence of biochemical events that follow irradiation. Images PMID:8419931

  19. The protein kinase homologue Ste20p is required to link the yeast pheromone response G-protein beta gamma subunits to downstream signalling components.

    PubMed Central

    Leberer, E; Dignard, D; Harcus, D; Thomas, D Y; Whiteway, M

    1992-01-01

    In the yeast Saccharomyces cerevisiae the G-protein beta gamma subunits have been shown to trigger downstream events of the pheromone response pathway. We have identified a new gene, designated STE20, which encodes a protein kinase homologue with sequence similarity to protein kinase C, which is required to transmit the pheromone signal from G beta gamma to downstream components of the signalling pathway. Overproduction of the kinase suppresses the mating defect of dominant-negative G beta mutations providing genetic evidence for an interaction with G beta, and epistasis experiments show that this kinase functions after or at the same point as G beta gamma, but before any of the other currently identified components of the signalling pathway. This points to a potentially new mechanism of G-protein mediated signal transduction, the activation of a protein kinase through G beta gamma. Images PMID:1464311

  20. Inhibition of T-cell antigen receptor-mediated transmembrane signaling by protein kinase C activation.

    PubMed Central

    Abraham, R T; Ho, S N; Barna, T J; Rusovick, K M; McKean, D J

    1988-01-01

    The murine T-lymphoma cell line LBRM-33 is known to require synergistic signals delivered through the antigen receptor (Ti-CD3) complex, together with interleukin 1 (IL-1), for activation of IL-2 gene expression and IL-2 production. Although 12-O-tetradecanoylphorbol-13-acetate (TPA) was capable of replacing IL-1 as an activating stimulus under certain conditions, biologic studies indicated that TPA failed to synergize with Ti-CD3-dependent stimuli under conditions in which IL-1 was clearly active. Acute exposure to TPA and other active phorbol esters resulted in a concentration-dependent inhibition of the increases in phosphoinositide hydrolysis and intracellular free Ca2+ concentration stimulated by phytohemagglutinin or anti-Ti antibodies. TPA treatment induced no direct alteration of phospholipase C enzymatic activities in LBRM-33 cells. In contrast, both Ti-CD3 cross-linkage and TPA rapidly stimulated the phosphorylation of identical CD3 complex polypeptides, presumably via activation of protein kinase C. Exposure of LBRM-33 cells to TPA resulted in a time-dependent, partial down-regulation of surface Ti-CD3 expression. Thus, TPA treatment inhibited the responsiveness of LBRM-33 cells to Ti-CD3-dependent stimuli by inducing an early desensitization of Ti-CD3 receptors, followed by a decrease in membrane receptor expression. These studies indicate that phorbol esters deliver bidirectional signals that both inhibit Ti-CD3-dependent phosphoinositide hydrolysis and augment IL-2 production in LBRM-33 cells. Images PMID:2977423

  1. Activin-Like Kinase 2 Functions in Peri-implantation Uterine Signaling in Mice and Humans

    PubMed Central

    Clementi, Caterina; Tripurani, Swamy K.; Large, Michael J.; Edson, Mark A.; Creighton, Chad J.; Hawkins, Shannon M.; Kovanci, Ertug; Kaartinen, Vesa; Lydon, John P.; Pangas, Stephanie A.; DeMayo, Francesco J.; Matzuk, Martin M.

    2013-01-01

    Implantation of a blastocyst in the uterus is a multistep process tightly controlled by an intricate regulatory network of interconnected ovarian, uterine, and embryonic factors. Bone morphogenetic protein (BMP) ligands and receptors are expressed in the uterus of pregnant mice, and BMP2 has been shown to be a key regulator of implantation. In this study, we investigated the roles of the BMP type 1 receptor, activin-like kinase 2 (ALK2), during mouse pregnancy by producing mice carrying a conditional ablation of Alk2 in the uterus (Alk2 cKO mice). In the absence of ALK2, embryos demonstrate delayed invasion into the uterine epithelium and stroma, and upon implantation, stromal cells fail to undergo uterine decidualization, resulting in sterility. Mechanistically, microarray analysis revealed that CCAAT/enhancer-binding protein β (Cebpb) expression is suppressed during decidualization in Alk2 cKO females. These findings and the similar phenotypes of Cebpb cKO and Alk2 cKO mice lead to the hypothesis that BMPs act upstream of CEBPB in the stroma to regulate decidualization. To test this hypothesis, we knocked down ALK2 in human uterine stromal cells (hESC) and discovered that ablation of ALK2 alters hESC decidualization and suppresses CEBPB mRNA and protein levels. Chromatin immunoprecipitation (ChIP) analysis of decidualizing hESC confirmed that BMP signaling proteins, SMAD1/5, directly regulate expression of CEBPB by binding a distinct regulatory sequence in the 3′ UTR of this gene; CEBPB, in turn, regulates the expression of progesterone receptor (PGR). Our work clarifies the conserved mechanisms through which BMPs regulate peri-implantation in rodents and primates and, for the first time, uncovers a linear pathway of BMP signaling through ALK2 to regulate CEBPB and, subsequently, PGR during decidualization. PMID:24244176

  2. Activation of Raf-1 and mitogen-activated protein kinase in murine macrophages partially mimics lipopolysaccharide-induced signaling events

    PubMed Central

    1995-01-01

    Lipopolysaccharide (LPS), a highly conserved component of the outer membrane of gram-negative bacteria, stimulates macrophages to release various cytokine and eicosanoid mediators of the immune response. The mechanism by which LPS stimulates these cells is poorly characterized. One of the most rapid LPS-stimulated events is the phosphorylation and activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinase. We wished to examine the role of MAP kinase in LPS- induced signaling in murine macrophages by activating MAP kinase independently of LPS. An expression vector encoding a Raf-1:estrogen receptor (ER) chimeric protein was transfected into the murine macrophage cell line RAW 264.7. Activation of this chimeric protein (delta Raf-1:ER) by estradiol resulted in rapid and prolonged activation of MAP kinase, as expected from previous results implicating Raf-1 as an upstream activator of this signaling cascade. LPS stimulation induced accumulation of MAP kinase phosphatase 1 messenger RNA, whereas delta Raf-1:ER activation did not, perhaps accounting for the more prolonged activation of MAP kinase seen in response to delta Raf-1:ER activation. Similarly, activation of DNA binding by the transcription factor, nuclear factor (NF) kappa B, as assessed by electrophoretic mobility shift assay, occurred in response to LPS stimulation but not in response to delta Raf-1:ER activation or phorbol myristate acetate (PMA) stimulation. Using an enzyme-linked immunosorbent assay for murine tumor necrosis factor alpha (TNF-alpha), we found that LPS and PMA stimulation and delta Raf-1:ER activation induced secretion of TNF-alpha, although the amount of TNF-alpha secreted in response to delta Raf-1:ER activation and PMA stimulation was approximately 20-fold less than that secreted in response to LPS. Correspondingly, accumulation of TNF-alpha messenger RNA was weakly induced by delta Raf-1:ER activation or PMA stimulation, whereas strong induction was noted in

  3. Activation of Raf-1 and mitogen-activated protein kinase in murine macrophages partially mimics lipopolysaccharide-induced signaling events.

    PubMed

    Hambleton, J; McMahon, M; DeFranco, A L

    1995-07-01

    Lipopolysaccharide (LPS), a highly conserved component of the outer membrane of gram-negative bacteria, stimulates macrophages to release various cytokine and eicosanoid mediators of the immune response. The mechanism by which LPS stimulates these cells is poorly characterized. One of the most rapid LPS-stimulated events is the phosphorylation and activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinase. We wished to examine the role of MAP kinase in LPS-induced signaling in murine macrophages by activating MAP kinase independently of LPS. An expression vector encoding a Raf-1:estrogen receptor (ER) chimeric protein was transfected into the murine macrophage cell line RAW 264.7. Activation of this chimeric protein (delta Raf-1:ER) by estradiol resulted in rapid and prolonged activation of MAP kinase, as expected from previous results implicating Raf-1 as an upstream activator of this signaling cascade. LPS stimulation induced accumulation of MAP kinase phosphatase 1 messenger RNA, whereas delta Raf-1:ER activation did not, perhaps accounting for the more prolonged activation of MAP kinase seen in response to delta Raf-1:ER activation. Similarly, activation of DNA binding by the transcription factor, nuclear factor (NF) kappa B, as assessed by electrophoretic mobility shift assay, occurred in response to LPS stimulation but not in response to delta Raf-1:ER activation or phorbol myristate acetate (PMA) stimulation. Using an enzyme-linked immunosorbent assay for murine tumor necrosis factor alpha (TNF-alpha), we found that LPS and PMA stimulation and delta Raf-1:ER activation induced secretion of TNF-alpha, although the amount of TNF-alpha secreted in response to delta Raf-1:ER activation and PMA stimulation was approximately 20-fold less than that secreted in response to LPS. Correspondingly, accumulation of TNF-alpha messenger RNA was weakly induced by delta Raf-1:ER activation or PMA stimulation, whereas strong induction was noted in

  4. Multiple regulatory domains on the Byr2 protein kinase.

    PubMed Central

    Tu, H; Barr, M; Dong, D L; Wigler, M

    1997-01-01

    Byr2 protein kinase, a homolog of mammalian mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEKK) and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation in the fission yeast Schizosaccharomyces pombe. Byr2 functions downstream of Ste4, Ras1, and the membrane-associated receptor-coupled heterotrimeric G-protein alpha subunit, Gpa1. Byr2 has a distinctive N-terminal kinase regulatory domain and a characteristic C-terminal kinase catalytic domain. Ste4 and Ras1 interact with the regulatory domain of Byr2 directly. Here, we define the domains of Byr2 that bind Ste4 and Ras1 and show that the Byr2 regulatory domain binds to the catalytic domain in the two-hybrid system. Using Byr2 mutants, we demonstrate that these direct physical interactions are all required for proper signaling. In particular, the physical association between Byr2 regulatory and catalytic domains appears to result in autoinhibition, the loss of which results in kinase activation. Furthermore, we provide evidence that Shk1, the S. pombe homolog of the STE20 protein kinase, can directly antagonize the Byr2 intramolecular interaction, possibly by phosphorylating Byr2. PMID:9315645

  5. Impaired signaling through the Fms-like tyrosine kinase 3 receptor increases osteoclast formation and bone damage in arthritis.

    PubMed

    Svensson, Mattias N D; Erlandsson, Malin C; Jonsson, Ing-Marie; Andersson, Karin M E; Bokarewa, Maria I

    2016-03-01

    Osteoclasts are bone-resorbing cells that accumulate in the joints of patients with rheumatoid arthritis causing severe bone damage. Fms-like tyrosine kinase 3 ligand is enriched in the synovial fluid of patients with rheumatoid arthritis, and local exposure to Fms-like tyrosine kinase 3 ligand aggravates arthritis in mice. Because Fms-like tyrosine kinase 3 ligand has been suggested to facilitate osteoclast differentiation, we asked whether Fms-like tyrosine kinase 3 ligand affects bone remodeling in arthritis. The effect of Fms-like tyrosine kinase 3 signaling on osteoclast development was studied by immunohistochemistry in methylated bovine serum albumin-induced arthritis using mice that lack the gene for Flt3l (Flt3L(-/-)) and by an in vitro assay. Bone and joint changes were studied morphologically and by microcomputer tomography. We found that Flt3L(-/-) mice had increased accumulations of osteoclasts in the periarticular area of the arthritic joint. This triggered bone destruction and trabecular bone loss. The increased number of osteoclasts in Flt3L(-/-) mice may be a consequence of insufficient expression of interferon regulatory factor 8. Treatment of Flt3L(-/-) mice with Fms-like tyrosine kinase 3 ligand increased expression of interferon regulatory factor 8, reduced the number of osteoclasts in arthritic mice, and promoted trabecular bone formation. Finally, the reduced number of regulatory T cells in the bone marrow of Flt3L(-/-) mice could further contribute to the increased osteoclastogenesis by reducing the ratio of regulatory T cells to T helper 17 cells. This study shows that Fms-like tyrosine kinase 3 ligand may serve as a negative regulator of osteoclast development by promoting transcription of interferon regulatory factor 8 and sustaining a balance between protective regulatory T cells and pathogenic T helper 17 cells in the pathogenesis of arthritis.

  6. The putative lipid transporter, Arv1, is required for activating pheromone-induced MAP kinase signaling in Saccharomyces cerevisiae.

    PubMed

    Villasmil, Michelle L; Ansbach, Alison; Nickels, Joseph T

    2011-02-01

    Saccharomyces cerevisiae haploid cells respond to extrinsic mating signals by forming polarized projections (shmoos), which are necessary for conjugation. We have examined the role of the putative lipid transporter, Arv1, in yeast mating, particularly the conserved Arv1 homology domain (AHD) within Arv1 and its role in this process. Previously it was shown that arv1 cells harbor defects in sphingolipid and glycosylphosphatidylinositol (GPI) biosyntheses and may harbor sterol trafficking defects. Here we demonstrate that arv1 cells are mating defective and cannot form shmoos. They lack the ability to initiate pheromone-induced G1 cell cycle arrest, due to failure to polarize PI(4,5)P(2) and the Ste5 scaffold, which results in weakened MAP kinase signaling activity. A mutant Ste5, Ste5(Q59L), which binds more tightly to the plasma membrane, suppresses the MAP kinase signaling defects of arv1 cells. Filipin staining shows arv1 cells contain altered levels of various sterol microdomains that persist throughout the mating process. Data suggest that the sterol trafficking defects of arv1 affect PI(4,5)P(2) polarization, which causes a mislocalization of Ste5, resulting in defective MAP kinase signaling and the inability to mate. Importantly, our studies show that the AHD of Arv1 is required for mating, pheromone-induced G1 cell cycle arrest, and for sterol trafficking.

  7. The Putative Lipid Transporter, Arv1, Is Required for Activating Pheromone-Induced MAP Kinase Signaling in Saccharomyces cerevisiae

    PubMed Central

    Villasmil, Michelle L.; Ansbach, Alison; Nickels, Joseph T.

    2011-01-01

    Saccharomyces cerevisiae haploid cells respond to extrinsic mating signals by forming polarized projections (shmoos), which are necessary for conjugation. We have examined the role of the putative lipid transporter, Arv1, in yeast mating, particularly the conserved Arv1 homology domain (AHD) within Arv1 and its role in this process. Previously it was shown that arv1 cells harbor defects in sphingolipid and glycosylphosphatidylinositol (GPI) biosyntheses and may harbor sterol trafficking defects. Here we demonstrate that arv1 cells are mating defective and cannot form shmoos. They lack the ability to initiate pheromone-induced G1 cell cycle arrest, due to failure to polarize PI(4,5)P2 and the Ste5 scaffold, which results in weakened MAP kinase signaling activity. A mutant Ste5, Ste5Q59L, which binds more tightly to the plasma membrane, suppresses the MAP kinase signaling defects of arv1 cells. Filipin staining shows arv1 cells contain altered levels of various sterol microdomains that persist throughout the mating process. Data suggest that the sterol trafficking defects of arv1 affect PI(4,5)P2 polarization, which causes a mislocalization of Ste5, resulting in defective MAP kinase signaling and the inability to mate. Importantly, our studies show that the AHD of Arv1 is required for mating, pheromone-induced G1 cell cycle arrest, and for sterol trafficking. PMID:21098723

  8. Signalling pathway leading to an activation of mitogen-activated protein kinase by stimulating M3 muscarinic receptor.

    PubMed

    Kim, J Y; Yang, M S; Oh, C D; Kim, K T; Ha, M J; Kang, S S; Chun, J S

    1999-01-15

    The signalling pathway leading to an activation of mitogen-activated protein (MAP) kinase subtypes Erk-1 and -2 upon stimulation of muscarinic receptor with carbachol in human neuroblastoma SK-N-BE2(C) cells was investigated. Carbachol activated Erk-1/-2 by stimulating M3 muscarinic receptor, as determined by specific antagonists for individual muscarinic receptors. The activation of Erk-1/-2 by carbachol was blocked by the inhibition or down-regulation of protein kinase C (PKC). Among the multiple PKC isoforms expressed in SK-N-BE2(C) cells, only PKCepsilon was activated by the treatment of carbachol, and selective down-regulation of PKCepsilon was sufficient to block Erk-1/-2 activation. Carbachol treatment induced activation of the serine/threonine protein kinase Raf, and an inhibition of Raf blocked Erk-1/-2 activation. Ectopic expression of inhibitory small GTPase Ras, RasN17, blocked the carbachol-induced Raf activation without affecting the activation of PKCepsilon, while the inhibition of PKC blocked the Raf activation. Thus, these results suggest that carbachol-induced activation of PKCepsilon mediates Erk-1/-2 activation by a sequential activation of Ras, Raf and MAP kinase kinase.

  9. The role of the Syk/Shp-1 kinase-phosphatase equilibrium in B cell development and signaling.

    PubMed

    Alsadeq, Ameera; Hobeika, Elias; Medgyesi, David; Kläsener, Kathrin; Reth, Michael

    2014-07-01

    Signal transduction from the BCR is regulated by the equilibrium between kinases (e.g., spleen tyrosine kinase [Syk]) and phosphatases (e.g., Shp-1). Previous studies showed that Syk-deficient B cells have a developmental block at the pro/pre-B cell stage, whereas a B cell-specific Shp-1 deficiency promoted B-1a cell development and led to autoimmunity. We generated B cell-specific Shp-1 and Syk double-knockout (DKO) mice and compared them to the single-knockout mice deficient for either Syk or Shp-1. Unlike Syk-deficient mice, the DKO mice can generate mature B cells, albeit at >20-fold reduced B cell numbers. The DKO B-2 cells are all Syk-negative, whereas the peritoneal B1 cells of the DKO mice still express Syk, indicating that they require this kinase for their proper development. The DKO B-2 cells cannot be stimulated via the BCR, whereas they are efficiently activated via TLR or CD40. We also found that in DKO pre-B cells, the kinase Zap70 is associated with the pre-BCR, suggesting that Zap70 is important to promote B cell maturation in the absence of Syk and SHP-1. Together, our data show that a properly balanced kinase/phosphatase equilibrium is crucial for normal B cell development and function.

  10. Inhibition of protein kinase Akt1 by apoptosis signal-regulating kinase-1 (ASK1) is involved in apoptotic inhibition of regulatory volume increase.

    PubMed

    Subramanyam, Muthangi; Takahashi, Nobuyuki; Hasegawa, Yuichi; Mohri, Tatsuma; Okada, Yasunobu

    2010-02-26

    Most animal cell types regulate their cell volume after an osmotic volume change. The regulatory volume increase (RVI) occurs through uptake of NaCl and osmotically obliged water after osmotic shrinkage. However, apoptotic cells undergo persistent cell shrinkage without showing signs of RVI. Persistence of the apoptotic volume decrease is a prerequisite to apoptosis induction. We previously demonstrated that volume regulation is inhibited in human epithelial HeLa cells stimulated with the apoptosis inducer. Here, we studied signaling mechanisms underlying the apoptotic inhibition of RVI in HeLa cells. Hypertonic stimulation was found to induce phosphorylation of a Ser/Thr protein kinase Akt (protein kinase B). Shrinkage-induced Akt activation was essential for RVI induction because RVI was suppressed by an Akt inhibitor, expression of a dominant negative form of Akt, or small interfering RNA-mediated knockdown of Akt1 (but not Akt2). Staurosporine, tumor necrosis factor-alpha, or a Fas ligand inhibited both RVI and hypertonicity-induced Akt activation in a manner sensitive to a scavenger for reactive oxygen species (ROS). Any of apoptosis inducers also induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) in a ROS-dependent manner. Suppression of (ASK1) expression blocked the effects of apoptosis, in hypertonic conditions, on both RVI induction and Akt activation. Thus, it is concluded that in human epithelial cells, shrinkage-induced activation of Akt1 is involved in the RVI process and that apoptotic inhibition of RVI is caused by inhibition of Akt activation, which results from ROS-mediated activation of ASK1. PMID:20048146

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

    SciTech Connect

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald

    2015-03-01

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

  12. T3-induced liver AMP-activated protein kinase signaling: Redox dependency and upregulation of downstream targets

    PubMed Central

    Videla, Luis A; Fernández, Virginia; Cornejo, Pamela; Vargas, Romina; Morales, Paula; Ceballo, Juan; Fischer, Alvaro; Escudero, Nicolás; Escobar, Oscar

    2014-01-01

    AIM: To investigate the redox dependency and promotion of downstream targets in thyroid hormone (T3)-induced AMP-activated protein kinase (AMPK) signaling as cellular energy sensor to limit metabolic stresses in the liver. METHODS: Fed male Sprague-Dawley rats were given a single ip dose of 0.1 mg T3/kg or T3 vehicle (NaOH 0.1 N; controls) and studied at 8 or 24 h after treatment. Separate groups of animals received 500 mg N-acetylcysteine (NAC)/kg or saline ip 30 min prior T3. Measurements included plasma and liver 8-isoprostane and serum β-hydroxybutyrate levels (ELISA), hepatic levels of mRNAs (qPCR), proteins (Western blot), and phosphorylated AMPK (ELISA). RESULTS: T3 upregulates AMPK signaling, including the upstream kinases Ca2+-calmodulin-dependent protein kinase kinase-β and transforming growth factor-β-activated kinase-1, with T3-induced reactive oxygen species having a causal role due to its suppression by pretreatment with the antioxidant NAC. Accordingly, AMPK targets acetyl-CoA carboxylase and cyclic AMP response element binding protein are phosphorylated, with the concomitant carnitine palmitoyltransferase-1α (CPT-1α) activation and higher expression of peroxisome proliferator-activated receptor-γ co-activator-1α and that of the fatty acid oxidation (FAO)-related enzymes CPT-1α, acyl-CoA oxidase 1, and acyl-CoA thioesterase 2. Under these conditions, T3 induced a significant increase in the serum levels of β-hydroxybutyrate, a surrogate marker for hepatic FAO. CONCLUSION: T3 administration activates liver AMPK signaling in a redox-dependent manner, leading to FAO enhancement as evidenced by the consequent ketogenic response, which may constitute a key molecular mechanism regulating energy dynamics to support T3 preconditioning against ischemia-reperfusion injury. PMID:25516653

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

    SciTech Connect

    Xie, Yuchao; Ramachandran, Anup; Breckenridge, David G.; Liles, John T.; Lebofsky, Margitta; Farhood, Anwar; Jaeschke, Hartmut

    2015-07-01

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

  14. The Tor and PKA signaling pathways independently target the Atg1/Atg13 protein kinase complex to control autophagy

    PubMed Central

    Stephan, Joseph S.; Yeh, Yuh-Ying; Ramachandran, Vidhya; Deminoff, Stephen J.; Herman, Paul K.

    2009-01-01

    Macroautophagy (or autophagy) is a conserved degradative pathway that has been implicated in a number of biological processes, including organismal aging, innate immunity, and the progression of human cancers. This pathway was initially identified as a cellular response to nutrient deprivation and is essential for cell survival during these periods of starvation. Autophagy is highly regulated and is under the control of a number of signaling pathways, including the Tor pathway, that coordinate cell growth with nutrient availability. These pathways appear to target a complex of proteins that contains the Atg1 protein kinase. The data here show that autophagy in Saccharomyces cerevisiae is also controlled by the cAMP-dependent protein kinase (PKA) pathway. Elevated levels of PKA activity inhibited autophagy and inactivation of the PKA pathway was sufficient to induce a robust autophagy response. We show that in addition to Atg1, PKA directly phosphorylates Atg13, a conserved regulator of Atg1 kinase activity. This phosphorylation regulates Atg13 localization to the preautophagosomal structure, the nucleation site from which autophagy pathway transport intermediates are formed. Atg13 is also phosphorylated in a Tor-dependent manner, but these modifications appear to occur at positions distinct from the PKA phosphorylation sites identified here. In all, our data indicate that the PKA and Tor pathways function independently to control autophagy in S. cerevisiae, and that the Atg1/Atg13 kinase complex is a key site of signal integration within this degradative pathway. PMID:19805182

  15. The Physarum polycephalum Genome Reveals Extensive Use of Prokaryotic Two-Component and Metazoan-Type Tyrosine Kinase Signaling

    PubMed Central

    Schaap, Pauline; Barrantes, Israel; Minx, Pat; Sasaki, Narie; Anderson, Roger W.; Bénard, Marianne; Biggar, Kyle K.; Buchler, Nicolas E.; Bundschuh, Ralf; Chen, Xiao; Fronick, Catrina; Fulton, Lucinda; Golderer, Georg; Jahn, Niels; Knoop, Volker; Landweber, Laura F.; Maric, Chrystelle; Miller, Dennis; Noegel, Angelika A.; Peace, Rob; Pierron, Gérard; Sasaki, Taeko; Schallenberg-Rüdinger, Mareike; Schleicher, Michael; Singh, Reema; Spaller, Thomas; Storey, Kenneth B.; Suzuki, Takamasa; Tomlinson, Chad; Tyson, John J.; Warren, Wesley C.; Werner, Ernst R.; Werner-Felmayer, Gabriele; Wilson, Richard K.; Winckler, Thomas; Gott, Jonatha M.; Glöckner, Gernot; Marwan, Wolfgang

    2016-01-01

    Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into early eukaryote evolution. We describe extensive use of histidine kinase-based two-component systems and tyrosine kinase signaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases in Acanthamoeba and Physarum as representatives of two distantly related subdivisions of Amoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfer. PMID:26615215

  16. Inhibition of either phosphatidylinositol 3-kinase/Akt or the mitogen/extracellular-regulated kinase, MEK/ERK, signaling pathways suppress growth of breast cancer cell lines, but MEK/ERK signaling is critical for cell survival.

    PubMed

    Ripple, Maureen O; Kalmadi, Sahana; Eastman, Alan

    2005-09-01

    The phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways are important integrators of growth and survival signals originating from extracellular stimuli. We assessed the importance of these signaling pathways in the growth and survival of 8 breast cell lines (MCF10A, an immortalized line; and 7 cancer cell lines). The cell lines expressed variable levels of both phosphorylated ERK and phosphorylated Akt, but these were unchanged by incubation in serum-free medium. Despite continued activity of these pathways, the cells arrested growth in the absence of serum demonstrating that additional pathways are required for growth. Incubation with the PI3K inhibitor LY294002 suppressed growth of all cell lines, but most remained viable for at least 7-14 days. This long-term survival may be attributable to recovery of phospho-Akt by 24-48 h despite the continued presence of active LY294002, suggesting that alternate pathways may be activating Akt. In contrast, incubation with the MEK inhibitor U0126 not only arrested growth, but also killed all the cell lines within 2-4 days in the absence of serum; the presence of serum only slighted extended viability, except in MCF10A and MDA-MB-468 cells, in which serum provided significantly greater protection. It is likely that these signaling pathways control the level of pro-and anti-apoptotic proteins, yet assessment of Bcl-2 and Bcl-X showed dramatic reduction in level only when large numbers of cells were dead suggesting this may be a consequence rather than cause of death. Overall, the results demonstrate that the MEK/ERK pathway represents the more critical pathway for cell survival of these breast cancer cell lines, and suggest this pathways represents the better target for cancer therapy.

  17. Momilactione B inhibits protein kinase A signaling and reduces tyrosinase-related proteins 1 and 2 expression in melanocytes.

    PubMed

    Lee, Ji Hae; Cho, Boram; Jun, Hee-jin; Seo, Woo-Duck; Kim, Dong-Woo; Cho, Kang-Jin; Lee, Sung-Joon

    2012-05-01

    Momilactone B (MB) is a terpenoid phytoalexin present in rice bran that exhibits several biological activities. MB reduced the melanin content in B16 melanocytes melanin content and inhibited tyrosinase activities. Using transcriptome analysis, the genes involved in protein kinase A (PKA) signaling were found to be markedly altered. B16 cells stimulated with MB had decreased concentrations of cAMP protein kinase A activity, and cAMP-response element-binding protein which is a key transcription factor for microphthalmia-associated transcription factor (MITF) expression. Accordingly, the expression of MITF and its target genes, which are essential for melanogenesis, were reduced. MB thus exhibits anti-melanogenic effects by repressing tyrosinase enzyme activity and inhibiting the PKA signaling pathway which, in turn, decreases melanogenic gene expression.

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

    PubMed

    Penela, P

    2016-01-01

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

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

    PubMed

    Penela, P

    2016-01-01

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

  20. Regulation of dopamine D2 receptor-mediated extracellular signal-regulated kinase signaling and spine formation by GABAA receptors in hippocampal neurons.

    PubMed

    Yoon, Dong-Hoon; Yoon, Sehyoun; Kim, Donghoon; Kim, Hyun; Baik, Ja-Hyun

    2015-01-23

    Dopamine (DA) signaling via DA receptors is known to control hippocampal activity that contributes to learning, memory, and synaptic plasticity. In primary hippocampal neuronal culture, we observed that dopamine D2 receptors (D2R) co-localized with certain subtypes of GABAA receptors, namely α1, β3, and γ2 subunits, as revealed by double immunofluorocytochemical analysis. Treatment with the D2R agonist, quinpirole, was shown to elicit an increase in phosphorylation of extracellular signal-regulated kinase (ERK) in hippocampal neurons. This phosphorylation was inhibited by pretreatment with the GABAA receptor agonist, muscimol. Furthermore, treatment of hippocampal neurons with quinpirole increased the dendritic spine density and this regulation was totally blocked by pretreatment with a MAP kinase kinase (MEK) inhibitor (PD98059), D2R antagonist (haloperidol), or by the GABAA receptor agonist, muscimol. These results suggest that D2R-mediated ERK phosphorylation can control spine formation and that the GABAA receptor negatively regulates the D2R-induced spine formation through ERK signaling in hippocampal neurons, thus indicating a potential role of D2R in the control of hippocampal neuronal excitability. PMID:25483619

  1. Regulation of extracellular-signal regulated kinase and c-Jun N-terminal kinase by G-protein-linked muscarinic acetylcholine receptors.

    PubMed Central

    Wylie, P G; Challiss, R A; Blank, J L

    1999-01-01

    Extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs, or stress-activated protein kinases) are activated by diverse extracellular signals and mediate a variety of cellular responses, including mitogenesis, differentiation, hypertrophy, inflammatory reactions and apoptosis. We have examined the involvement of Ca2+ and protein kinase C (PKC) in ERK and JNK activation by the human G-protein-coupled m2 and m3 muscarinic acetylcholine receptors (mAChR) expressed in Chinese hamster ovary (CHO) cells. We show that the Ca2+-mobilizing m3 AChR is efficiently coupled to JNK and ERK activation, whereas the m2 AChR activates ERK but not JNK. Activation of JNK in CHO-m3 cells by the agonist methacholine (MCh) was delayed in onset and more sustained relative to that of ERK in either CHO-m2 or CHO-m3 cells. The EC50 values for MCh-induced ERK activation in both cell types were essentially identical and similar to that for JNK activation in CHO-m3 cells, suggesting little amplification of the response. Agonist-stimulated Ins(1,4,5)P3 accumulation in CHO-m3 cells was insensitive to pertussis toxin (PTX), consistent with a Gq/phosphoinositide-specific phospholipase C-beta mediated pathway, whereas a significant component of ERK and JNK activation in CHO-m3 cells was PTX-sensitive, indicating Gi/o involvement. Using manipulations that prevent receptor-mediated extracellular Ca2+ influx and intracellular Ca2+-store release, we also show that ERK activation by m2 and m3 receptors is Ca2+-independent. In contrast, a significant component (>50%) of JNK activation mediated by the m3 AChR was dependent on Ca2+, mainly derived from extracellular influx. PKC inhibition and down-regulation studies suggested that JNK activation was negatively regulated by PKC. Conversely, ERK activation by both m2 and m3 AChRs required PKC, suggesting a novel mechanism for PKC activation by PTX-sensitive m2 AChRs. In summary, mAChRs activate JNK and ERK via divergent mechanisms

  2. Tyrosine Kinase 2-mediated Signal Transduction in T Lymphocytes Is Blocked by Pharmacological Stabilization of Its Pseudokinase Domain*

    PubMed Central

    Tokarski, John S.; Zupa-Fernandez, Adriana; Tredup, Jeffrey A.; Pike, Kristen; Chang, ChiehYing; Xie, Dianlin; Cheng, Lihong; Pedicord, Donna; Muckelbauer, Jodi; Johnson, Stephen R.; Wu, Sophie; Edavettal, Suzanne C.; Hong, Yang; Witmer, Mark R.; Elkin, Lisa L.; Blat, Yuval; Pitts, William J.; Weinstein, David S.; Burke, James R.

    2015-01-01

    Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing approach to the treatment of autoimmunity. Using a chemogenomics approach marrying kinome-wide inhibitory profiles of a compound library with the cellular activity against an IL-23-stimulated transcriptional response in T lymphocytes, a class of inhibitors was identified that bind to and stabilize the pseudokinase domain of the Janus kinase tyrosine kinase 2 (Tyk2), resulting in blockade of receptor-mediated activation of the adjacent catalytic domain. These Tyk2 pseudokinase domain stabilizers were also shown to inhibit Tyk2-dependent signaling through the Type I interferon receptor but not Tyk2-independent signaling and transcriptional cellular assays, including stimulation through the receptors for IL-2 (JAK1- and JAK3-dependent) and thrombopoietin (JAK2-dependent), demonstrating the high functional selectivity of this approach. A crystal structure of the pseudokinase domain liganded with a representative example showed the compound bound to a site analogous to the ATP-binding site in catalytic kinases with features consistent with high ligand selectivity. The results support a model where the pseudokinase domain regulates activation of the catalytic domain by forming receptor-regulated inhibitory interactions. Tyk2 pseudokinase stabilizers, therefore, represent a novel approach to the design of potent and selective agents for the treatment of autoimmunity. PMID:25762719

  3. The Brucella abortus virulence regulator, LovhK, is a sensor kinase in the general stress response signalling pathway.

    PubMed

    Kim, Hye-Sook; Willett, Jonathan W; Jain-Gupta, Neeta; Fiebig, Aretha; Crosson, Sean

    2014-11-01

    In the intracellular pathogen Brucella abortus, the general stress response (GSR) signalling system determines survival under acute stress conditions in vitro, and is required for long-term residence in a mammalian host. To date, the identity of the Brucella sensor kinase(s) that function to perceive stress and directly activate GSR signalling have remained undefined. We demonstrate that the flavin-binding sensor histidine kinase, LovhK (bab2_0652), functions as a primary B. abortus GSR sensor. LovhK rapidly and specifically phosphorylates the central GSR regulator, PhyR, and activates transcription of a set of genes that closely overlaps the known B. abortus GSR regulon. Deletion of lovhK severely compromises cell survival under defined oxidative and acid stress conditions. We further show that lovhK is required for cell survival during the early phase of mammalian cell infection and for establishment of long-term residence in a mouse infection model. Finally, we present evidence that particular regions of primary structure within the two N-terminal PAS domains of LovhK have distinct sensory roles under specific environmental conditions. This study elucidates new molecular components of a conserved signalling pathway that regulates B. abortus stress physiology and infection biology.

  4. The Brucella abortus virulence regulator, LovhK, is a sensor kinase in the general stress response signaling pathway

    PubMed Central

    Kim, Hye-Sook; Willett, Jonathan W.; Jain-Gupta, Neeta; Fiebig, Aretha; Crosson, Sean

    2014-01-01

    Summary In the intracellular pathogen Brucella abortus, the general stress response (GSR) signaling system determines survival under acute stress conditions in vitro, and is required for long-term residence in a mammalian host. To date, the identity of the Brucella sensor kinase(s) that function to perceive stress and directly activate GSR signaling have remained undefined. We demonstrate that the flavin-binding sensor histidine kinase, LovhK (bab2_0652), functions as a primary B. abortus GSR sensor. LovhK efficiently and specifically phosphorylates the central GSR regulator, PhyR, and activates transcription of a set of genes that closely overlaps the known B. abortus GSR regulon. Deletion of lovhK severely compromises cell survival under defined oxidative and acid stress conditions. We further show that lovhK is required for cell survival during the early phase of mammalian cell infection and for establishment of long-term residence in a mouse infection model. Finally, we present evidence that particular regions of primary structure within the two N-terminal PAS domains of LovhK have distinct sensory roles under specific environmental conditions. This study elucidates new molecular components of a conserved signaling pathway that regulates B. abortus stress physiology and infection biology. PMID:25257300

  5. Homocysteine-NMDA receptor mediated activation of extracellular-signal regulated kinase leads to neuronal cell death

    PubMed Central

    Poddar, Ranjana; Paul, Surojit

    2009-01-01

    Hyper-homocysteinemia is an independent risk factor for stroke and neurological abnormalities. However the underlying cellular mechanisms by which elevated homocysteine can promote neuronal death is not clear. In the present study we have examined the role of NMDA receptor mediated activation of the extracellular-signal regulated mitogen activated protein (ERK MAP) kinase pathway in homocysteine-dependent neurotoxicity. The study demonstrates that in neurons L-homocysteine-induced cell death is mediated through activation of NMDA receptors. The study also shows that homocysteine-dependent NMDA receptor stimulation and resultant Ca2+ influx leads to rapid and sustained phosphorylation of ERK MAP kinase. Inhibition of ERK phosphorylation attenuates homocysteine mediated neuronal cell death thereby demonstrating that activation of ERK MAP kinase signaling pathway is an intermediate step that couples homocysteine mediated NMDA receptor stimulation to neuronal death. The findings also show that cAMP response-element binding protein (CREB), a pro-survival transcription factor and a downstream target of ERK, is only transiently activated following homocysteine exposure. The sustained activation of ERK but a transient activation of CREB together suggest that exposure to homocysteine initiates a feedback loop that shuts off CREB signaling without affecting ERK phosphorylation and thereby facilitates homocysteine mediated neurotoxicity. PMID:19508427

  6. TRPC3 amplifies B-cell receptor-induced ERK signalling via protein kinase D-dependent Rap1 activation.

    PubMed

    Numaga-Tomita, Takuro; Nishida, Motohiro; Putney, James W; Mori, Yasuo

    2016-01-15

    Sustained activation of extracellular-signal-regulated kinase (ERK) has an important role in the decision regarding the cell fate of B-lymphocytes. Recently, we demonstrated that the diacylglycerol-activated non-selective cation channel canonical transient receptor potential 3 (TRPC3) is required for the sustained ERK activation induced by the B-cell receptor. However, the signalling mechanism underlying TRPC3-mediated ERK activation remains elusive. In the present study, we have shown that TRPC3 mediates Ca(2+) influx to sustain activation of protein kinase D (PKD) in a protein kinase C-dependent manner in DT40 B-lymphocytes. The later phase of ERK activation depends on the small G-protein Rap1, known as a downstream target of PKD, whereas the earlier phase of ERK activation depends on the Ras protein. It is of interest that sustained ERK phosphorylation is required for the full induction of the immediate early gene Egr-1 (early growth response 1). These results suggest that TRPC3 reorganizes the BCR signalling complex by switching the subtype of small G-proteins to sustain ERK activation in B-lymphocytes.

  7. Mitogen-activated protein kinase/extracellular signal-regulated kinase 2 regulates cytoskeletal organization and chemotaxis via catalytic and microtubule-specific interactions.

    PubMed Central

    Reszka, A A; Bulinski, J C; Krebs, E G; Fischer, E H

    1997-01-01

    The extracellular signal-regulated kinases (ERKs) 1 and 2 are mitogen-activated protein kinases that act as key components in a signaling cascade linking growth factor receptors to the cytoskeleton and the nucleus. ERK2 mutants have been used to alter cytoskeletal regulation in Chinese hamster ovary cells without affecting cell growth or feedback signaling. Mutation of the unique loop L6 (residues 91-95), which is in a portion of the molecule that is cryptic upon the binding of ERK2 to the microtubules (MTs), generated significant morphological alterations. Most notable phenotypes were observed after expression of a combined mutant incorporating changes to both L6 and the TEY phosphorylation lip, including a 70% increase in cell spreading. Actin stress fibers in these cells, which normally formed a single broad parallel array, were arranged in three or more orientations or in fan-like arrays. MTs, which ordinarily extend longitudinally from the centrosome, spread radially, covering a larger surface area. Single, but not the double, mutations of the Thr and Tyr residues of the TEY phosphorylation lip caused a ca. 25% increase in cell spreading, accompanied by a threefold increase in chemotactic cell migration. Mutation of Lys-52 triggered a 48% increase in cell spreading but no alteration to chemotaxis. These findings suggest that wild-type ERK2 inhibits the organization of the cytoskeleton, the spreading of the cell, and chemotactic migration. This involves control of the orientation of actin and MTs and the positioning of focal adhesions via regulatory interactions that may occur on the MTs. Images PMID:9243503

  8. Signals controlling un-differentiated states in embryonic stem and cancer cells: role of the phosphatidylinositol 3' kinase pathway.

    PubMed

    Voskas, Daniel; Ling, Ling Sunny; Woodgett, James Robert

    2014-10-01

    The capacity of embryonic stem (ES) cells to differentiate into cell lineages comprising the three germ layers makes them powerful tools for studying mammalian early embryonic development in vitro. The human body consists of approximately 210 different somatic cell types, the majority of which have limited proliferative capacity. However, both stem cells and cancer cells bypass this replicative barrier and undergo symmetric division indefinitely when cultured under defined conditions. Several signal transduction pathways play important roles in regulating stem cell development, and aberrant expression of components of these pathways is linked to cancer. Among signaling systems, the critical role of leukemia inhibitory factor (LIF) coupled to the Jak/STAT3 (signal transduction and activation of transcription-3) pathway in maintaining stem cell self-renewal has been extensively reviewed. This pathway additionally plays multiple roles in tumorigenesis. Likewise, the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway has been determined to play an important role in both stem cell maintenance and tumor development. This pathway is often induced in cancer with frequent mutational activation of the catalytic subunit of PI3K or loss of a primary PI3K antagonist, phosphatase and tensin homolog deleted on chromosome ten (PTEN). This review focusses on roles of the PI3K signal transduction pathway components, with emphasis on functions in stem cell maintenance and cancer. Since the PI3K pathway impinges on and collaborates with other signaling pathways in regulating stem cell development and/or cancer, aspects of the canonical Wnt, Ras/mitogen-activated protein kinase (MAPK), and TGF-β signaling pathways are also discussed.

  9. Silver Nanoparticles Disrupt GDNF/Fyn kinase Signaling in Spermatogonial Stem Cells

    PubMed Central

    Braydich-Stolle, Laura K.; Lucas, Benjamin; Schrand, Amanda; Murdock, Richard C.; Lee, Timothy; Schlager, John J.; Hussain, Saber M.; Hofmann, Marie-Claude

    2010-01-01

    Silver nanoparticles (Ag-NPs) are being utilized in an increasing number of fields and are components of antibacterial coatings, antistatic materials, superconductors, and biosensors. A number of reports have now described the toxic effects of silver nanoparticles on somatic cells; however, no study has examined their effects on the germ line at the molecular level. Spermatogenesis is a complex biological process that is particularly sensitive to environmental insults. Many chemicals, including ultrafine particles, have a negative effect on the germ line, either by directly affecting the germ cells or by indirectly acting on the somatic cells of the testis. In the present study, we have assessed the impact of different doses of Ag-NPs, as well as their size and biocompatible coating, on the proliferation of mouse spermatogonial stem cells (SSCs), which are at the origin of the germ line in the adult testis. At concentrations ≥ 10 μg/ml, Ag-NPs induced a significant decline in SSCs proliferation, which was also dependent on their size and coating. At the concentration of 10 μg/ml, reactive oxygen species production and/or apoptosis did not seem to play a major role; therefore, we explored other mechanisms to explain the decrease in cell proliferation. Because glial cell line–derived neurotrophic factor (GDNF) is vital for SSC self-renewal in vitro and in vivo, we evaluated the effects of Ag-NPs on GDNF-mediated signaling in these cells. Although the nanoparticles did not reduce GDNF binding or Ret receptor activity, our data revealed that already at a concentration of 10 μg/ml, silver nanoparticles specifically interact with Fyn kinase downstream of Ret and impair SSC proliferation in vitro. In addition, we demonstrated that the particle coating was degraded upon interaction with the intracellular microenvironment, reducing biocompatibility. PMID:20488942