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1
Adams, Deanna Grace. "Characterization of the stress-activated protein kinase, MEKK3." Diss., The University of Arizona, 2002. http://hdl.handle.net/10150/279970.
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The cAMP-dependent protein kinase, PKA, mediates diverse cellular processes including proliferation and differentiation, as well as the phosphorylation of proteins involved in cell death and survival such as BAD and GSK-3beta. In this work, I demonstrate that PKA phosphorylates the stress-activated protein kinase, MEKK3 in vivo and in vitro. When (His)6FLAG·MEKK3 was expressed in Sf9 insect cells and purified with Ni-Sepharose, we identified 14-3-3 protein by liquid chromatography and electrospray tandem mass spectrometry (LC-MS) as co-purifying with recombinant MEKK3. The yeast two hybrid system was also utilized to identify 14-3-3 as interacting proteins with MEKK3 as well as Bcl-xL. The interaction between MEKK3 and Bcl-xL was specific for the caspase-cleaved Bcl-x L which suggests a role for MEKK3 in apoptotic signaling pathways. However, the physiological significance of this interaction is unclear since caspase-cleaved Bcl-xL did not act as a MEKK3 substrate or alter MEKK3 kinase activity. Since 14-3-3 proteins have been reported to interact with proteins through phosphoserine, we sequenced (His)6FLAG·MEKK3 by LC-MS to identify phosphorylated amino acids. Of the tryptic peptides sequenced, two consisted of amino acids 164--174 and 335--349 and serines 166 and 337 were phosphorylated within the respective peptides. Phosphorylation of both serines was localized within two consensus PKA phosphorylation sites, RXX(S/T). PKA activators such as serum and forskolin increased phosphorylation of endogenous MEKK3 at Ser166 as well as the preferential recruitment of phospho-MEKK3 with 14-3-3. These results connect the stress-activated pathways regulated by MEKK3 with pathways regulated by PKA.
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Brancho, Deborah Marie. "Regulation and Function of Stress-Activated Protein Kinase Signal Transduction Pathways: A Dissertation." eScholarship@UMMS, 2005. https://escholarship.umassmed.edu/gsbs_diss/101.
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The c-Jun NH2-terminal kinase (JNK) group and the p38 group of mitogen-activated protein kinases (MAPK) are stress-activated protein kinases that regulate cell proliferation, differentiation, development, and apoptosis. These protein kinases are involved in a signal transduction cascade that includes a MAP kinase (MAPK), a MAP kinase kinase (MAP2K), and a MAP kinase kinase kinase (MAP3K). MAPK are phosphorylated and activated by the MAP2K, which are phosphorylated and activated by various MAP3K.
The work presented in this dissertation focuses on understanding the regulation and function of the JNK and p38 MAPK pathways. Two different strategies were utilized. First, I used molecular and biochemical techniques to examine how MAP2K and MAP3K mediate signaling specificity and to define their role in the MAPK pathway. Second, I used gene targeted disruption studies to determine the in vivo role ofMAP2K and MAP3K in MAPK activation. I specifically used these approaches to examine: (1) docking interactions between p38 MAPK and MAP2K [MKK3 and MKK6 (Chapter II)]; (2) the differential activation of p38 MAPK by MAP2K [MKK3, MKK4, and MKK6 (Chapter III)]; and (3) the selective involvement of the mixed lineage kinase (MLK) group of MAP3K in JNK and p38 MAPK activation (Chapter IV and Appendix). In addition, I analyzed the role of the MKK3 and MKK6 MAP2K in cell proliferation and the role of the MLK MAP3K in adipocyte differentiation (Chapter III and Chapter IV). Together, these data provide insight into the regulation and function of the stress-activated MAPK signal transduction pathways.
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Ferreiro, Neira Isabel. "Transcriptional regulation by the mammalian stress-activated protein kinase p38." Doctoral thesis, Universitat Pompeu Fabra, 2011. http://hdl.handle.net/10803/80661.
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Regulation of transcription by Stress Activated Protein Kinases (SAPKs) is an essential aspect for adaptation to extracellular stimuli. In mammals, the activation of the p38 SAPK results in the regulation of gene expression through the direct phosphorylation of several transcription factors. However, how p38 SAPK regulates the proper gene expression program of adaptation to stress as well as the basic mechanisms used by the SAPK remains uncharacterized. The results displayed in this manuscript show that the p38 SAPK plays a central role in the regulation of gene expression upon stress, as up to 80% of the upregulated genes are p38 SAPK dependent. Moreover, we also observed that a specific set of genes were upregulated in response to each specific stimuli, and just a small set of genes were commonly up-regulated by several stresses, which involves mainly transcription factors. In addition, we observed that, to proper regulate gene transcription, the p38 SAPK is recruited to stress-induced promoters via its interaction with transcription factors. Additionally, p38 activity allows the recruitment of RNA polymerase II and the MAPKK MKK6 to stress-responsive promoters. The presence of active p38 SAPK at open reading frames also suggests the involvement of the SAPK in elongation. Altogether, the results showed in this manuscript establish the p38 SAPK as an essential regulator in the transcriptional response to stress, as well as define new roles for p38 in the regulation of transcription in response to stress.La regulación de la transcripción por las Proteínas Quinasas activadas por Estrés (SAPKs) es un aspecto esencial para la adaptación a los estímulos extracelulares. En mamíferos, la activación de la SAPK p38 da lugar a la regulación de la expresión génica a través de la fosforilación de varios factores de transcripción. Sin embargo, cómo p38 SAPK regula el programa de expresión génica de adaptación al estrés así como los mecanismos utilizados por la SAPK permanece sin caracterizar. Los resultados presentados en este manuscrito muestran que p38 SAPK juega un rol central en la regulación de la expresión génica en respuesta a estrés, ya que hasta el 80% de los genes inducidos son dependientes de p38 SAPK. También observamos que en respuesta a cada tipo de estrés se induce un grupo de genes específicos, y sólo hay una pequeña respuesta de genes comunes a los diferentes tipos de estrés la cual engloba principalmente factores de transcripción. Además, hemos observado que para regular la transcripción, p38 se recluta a los promotores de respuesta a estrés a través de su interacción con factores de transcripción. Asimismo, la actividad de p38 permite el reclutamiento de la RNA Polimerasa II y de la MAPKK MKK6 a los promotores inducidos por estrés. La presencia de p38 activa en las regiones codificantes sugiere su participación durante la elongación. En conjunto, los resultados mostrados en este manuscrito establecen a p38 como un regulador esencial de la transcripción en respuesta a estrés, así como definen nuevas funciones de p38 en la regulación de la transcripción en respuesta a estrés.
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Auciello, Francesca Romana. "Canonical and non-canonical regulation of AMP-activated protein kinase." Thesis, University of Dundee, 2015. https://discovery.dundee.ac.uk/en/studentTheses/2720a2b7-3f1e-445c-b008-c5c235f35395.
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The AMP-activated protein kinase (AMPK) is a sensor of cellular energy stress that, once activated, promotes ATP-producing process while it switches off ATP-consuming pathways, in order to restore the cellular energetic balance under conditions of stress. Activation of AMPK is dependent on the phosphorylation of the residue Thr172 in its α subunit. This phosphorylation is generally mediated by the known tumour suppressor LKB1, but also CaMKKβ has been shown to phosphorylate AMPK. As its name suggests, AMPK is also activated by the binding of AMP to its γ subunit. This binding causes a >10 fold allosteric stimulation, promotes phosphorylation of Thr172 by upstream kinases and protects AMPK from dephosphorylation of Thr172 by protein phosphatase(s). In 2010 it was reported that oxidative stress mediated by H2O2 activated AMPK by increasing the cellular AMP:ATP and ADP:ATP ratios (Hawley et al, 2010). However, the same year another work suggested that the mechanism of activation of AMPK by H2O2 was direct, independent of AMP and involved the oxidation of two cysteine residues in the α subunit of AMPK (Zmijewski et al, 2010). Given this discrepancy, here we provided evidence that H2O2, generated by addition of glucose oxidase in the cell medium, activates AMPK mostly through an increase of AMP:ATP and ADP:ATP ratios, as previously suggested in our laboratory. However, it seems that there might be a second, minor mechanism of activation that is independent of the changes in cellular nucleotides. This second mechanism was not identified in our previous work because we were not aware of how rapidly a single bolus of H2O2 can be metabolized by the antioxidant defences of the cell. We could not identify the alternative mechanism of activation by H2O2 but showed that H2O2 could protect Thr172 from dephosphorylation, which might suggest a direct effect of H2O2 on the phosphatase(s) dephosphorylating AMPK. However, since the identity of this phosphatase(s) remains unclear, we could not rule out the possibility that the protection from dephosphorylation that we observed could still be mediated by the increase in AMP:ATP and ADP:ATP ratios. Moreover, it remains still possible that a direct effect of H2O2 on AMPK might be responsible for the small but significant activation we detected in cell expressing a nucleotides-insensitive mutant of AMPK. Recently, a new crystal structure of AMPK obtained by Xiao et al (2013) provided new insights about AMPK structure and regulation. In particular, the authors identified a new binding pocket located at the interface between the N-lobe of the α-kinase domain and the β-CBM of AMPK, which appeared to be the binding site for two direct activators of AMPK: A769662 and 991. Here we confirm that this novel binding pocket is indeed the binding site for both A769662 and 991, and provide evidence that another direct activator of AMPK, MT63-78, also binds at the same site. Mutation of two important residues in this pocket (Lys29 and Lys31 of the α2 subunit) abolished the allosteric stimulation of AMPK by A769662, 991 and MT63-78 while it had no effect on allosteric stimulation by AMP. However, we also showed that the same mutation abolished protection against Thr172 dephosphorylation not only by A769662, 991 and MT63-78, but also by phenformin and H2O2, which are known to activate AMPK by increasing the AMP:ATP and ADP:ATP ratios. These data show that the integrity of this pocket is important for the effect of AMP to protect against Thr172 dephosphorylation, but not for its ability to cause allosteric stimulation. Moreover, in HEK-293 cell stably expressing an α2 subunit carrying the mutation of both Lys29 and Lys31, the basal activity of AMPK due to Thr172 phosphorylation was almost 6-fold less than in cells expressing wild-type α2. This result pointed out for the first time that there might be a natural ligand binding in the newly discovered binding pocket that is not able to bind to the double mutant, explaining the difference in activity observed. However the identity of this possible natural ligand remains unclear and more studies will be necessary to uncover it.
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Foltz, Ian Nevin. "Regulation of the stress-activated protein kinase pathways in hematopoietic cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ38887.pdf.
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O'Ferrall, Erin K. "Phosphorylation of the neurofilament heavy subunit by stress-activated protein kinase." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=33433.
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Stress-activated protein kinases (SAPKs) were previously implicated in the phosphorylation of the neurofilament heavy subunit (NFH). This study presents direct evidence that stress-induced phosphorylation of NFH in both differentiated PC12 cells and cultured sensory neurons is inhibited by CEP-1347 (KT7515), a specific inhibitor of SAPK activation. In addition, long-term treatment of unstressed sensory neurons with CEP-1347 decreased the phosphorylation state of NFH in neurites. CEP-1347 differentially inhibited the activation of various SAPK isoforms in neuronal cell body and neurite fractions. Specifically, activation of a 55 kDa SAPK isoform in the neurite fraction was highly sensitive to CEP-1347 inhibition, with an IC50 of about 0.02 muM, while IC50 values for other SAPK species were at least 10-fold higher. The data indicate that SAPKs are involved in both constitutive phosphorylation of axonal NFH and stress-induced phosphorylation of perikaryal NFH in cultured sensory neurons and that SAPKs in the two neuronal compartments are activated by different signaling pathways.
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De, Zutter Gerard S. "Stress Activated Protein Kinase Regulation of Gene Expression in Apoptotic Neurons: A Dissertation." eScholarship@UMMS, 2001. https://escholarship.umassmed.edu/gsbs_diss/168.
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Summary
Basic biological processes require gene expression. Tightly regulated molecules known as transcription factors mediate the expression of genes in development and disease. Signal transduction pathways, which respond to environmental cues or stressors are major regulators of the transcription factors. Use of macromolecular synthesis inhibitors in models of normal neurodevelopment and neurodegenerative cell death has led to the discovery that gene expression is required for these processes to occur (Martin et. al.,(1988), J Cell Biol 106p829). To date, however, the identities of very few of the genes required in these events have been revealed. Hence, the activation or requirement of specific signaling pathways leading to the expression of known apoptotic genes is not well established. Utilizing the neurothrophic factor deprivation and neurotoxin models of programmed cell death we address these gaps in our understanding of the molecular mechanism of apoptosis as it occurs in neuronal cell death.
Nerve growth factor (NGF) withdrawal from PC12 cells leads to the activation of p38 and apoptosis. The functional significance of 38 activation in this paradigm of cell death is not known. To increase our understanding of apoptosis I examined the requirement for p38 activity in pro-apoptotic gene expression in PC12 cells. I performed a subtractive hybridization that led to the identification of the monoamine oxidase (MAG) gene as induced in response to NGF withdrawal. Using the p38 inhibitor PD169316 I showed that the NGF withdrawal stimulated induction of the MAG gene and apoptosis is blocked by inhibition of the p38 MAP kinase pathway. I also determined that the MAG inhibitor clorgyline blocked cell death indicating that MAG activity contributes to the cell death caused by NGF withdrawal. Together, these data indicate that the p38 MAP kinase pathway targets the MAG gene in response to apoptotic stimuli.
To study the requirement for the JNK signaling pathway in neurodegeneration I stimulated primary cortical neurons with the neurotoxin arsenite. Arsenite treatment of primary neurons leads to both JNK and p38 activation and subsequently apoptosis. Utilizing transgenic mice lacking the JNK3 gene I demonstrated that JNK3 specifically contributes to the effects of arsenite in these cells. Ribonuclease protection assays were used to identify Fas ligand as a molecule whose arsenite-induced expression is dependent on the JNK3 signal transduction pathway. Furthermore, I have shown that neurons deficient in signaling mediated by the receptor for Fas ligand are resistant to cell death due to arsenite treatment. These results in total have established that the JNK3 mediated expression of Fas ligand contributes to the arsenite induced death of cortical neurons.
In summary, the work presented in these studies identifies the JNK and p38 MAP kinase signal transduction pathways as mediators of apoptosis in neuronal cells. Importantly, I have provided evidence that these stress activated pathways are responsible for the expression of specific genes in apoptotic neuronal cells.
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Cheetham, Jill. "The Regulation of the Hog1 Stress Activated Protein Kinase in Candida albicans." Thesis, University of Newcastle Upon Tyne, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.489302.
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Yates, Alexandra Caroline. "Stress-activated protein kinases and neurodegenerative disease." Thesis, King's College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.287325.
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Schroder, Wayne Ashley. "Cloning and Characterisation of the Human SinRIP Proteins." Thesis, Griffith University, 2003. http://hdl.handle.net/10072/366190.
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This thesis describes the cloning and characterisation of a novel human gene and its protein products, which have been designated SAPK- and Ras-interacting protein (SinRIP). SinRIP shares identity with JC310, a partial human cDNA that was previously identified a candidate Ras-inhibitor (Colicelli et al., 1991, Proc Natl Acad Sci USA 88, p. 2913). In this study, it was shown that SinRIP is a member of an orthologous family of proteins that is conserved from yeast to mammals and contains proteins involved in Ras- and SAPK-mediated signalling pathways. Comparison of this family of proteins showed that human SinRIP contains a potential Ras-binding domain (RBD; residues 279-354), a PH-like domain (PHL; 376-487), and a highly conserved novel region designated the CRIM (134-265). Several other potential targeting sites, such as nuclear localisation signals and target sites for kinases, were identified within the SinRIP sequence. The human SinRIP gene is unusually large (>280 kbp) and is located on chromosome 9 at 9q34. SinRIP mRNA was detected in a wide variety of tissue-types and cell lines by RT-PCR, and the SinRIP sequences in the EST database were derived from an diverse array of tissues, suggesting a widespread or ubiquitous expression. Northern blot analysis revealed the highest levels in skeletal muscle and heart tissue. However, the steady-state levels of SinRIP mRNA vary greatly from cell to cell, and SinRIP expression is likely to be regulated at multiple post-transcriptional levels. It was shown that SinRIP mRNA is likely to be translated inefficiently by the normal cap-scanning mechanism, due to the presence of a GC-rich and structured 5’-UTR, which also contains upstream ORFs. Alternative polyadenylation signals in the SinRIP 3’-UTR can be used, resulting in the expression of short and long SinRIP mRNA isoforms. Several potential A/T-rich regulatory elements were also identified in SinRIP mRNA, which may target specific SinRIP mRNA isoforms for rapid degradation. Importantly, it was shown that SinRIP mRNA is alternatively spliced, resulting in the production of distinct SinRIP protein isoforms. Three isoforms, SinRIP2-4, were definitively identified by RT-PCR and full-length cloning. The SinRIP isoforms contain deletions in conserved regions, and are likely to have biochemical characteristics that are different to full-length SinRIP1. SinRIP2 is C-terminally truncated and lacks the PHL domain and part of the RBD, and relatively high levels of SinRIP2 expression arelikely to occur in kidneys. The RBD is disrupted in SinRIP3, but all other domains are intact, and RT-PCR analyses suggest that SinRIP3 is present in some cells at levels comparable to SinRIP1. A rabbit polyclonal antiserum against SinRIP was generated and detected endogenous SinRIP proteins. Using the anti-SinRIP antibody in immunoblots, multiple SinRIP isoforms were observed in most cell types. SinRIP1 and another endogenous SinRIP protein, likely to be SinRIP3, were detected in most cell lines, and appear to be are the major SinRIP proteins expressed in most cells. The subcellular localisation of both recombinant and endogenous SinRIP proteins was investigated by immunofluorescence assays and biochemical fractionation. Recombinant SinRIP1 protein was found in the cytoplasm and associated with the plasma membrane. In contrast, the SinRIP2 protein was predominantly nuclear, with only low-level cytoplasmic staining observed. The endogenous SinRIP proteins, likely to comprise these and other SinRIP isoforms, were found in both the nucleus and cytoplasm. SinRIP1 interacted with GTP-bound (active) Ras, but not GDP-bound (inactive) Ras, in an in vitro assay, and also co-localised with activated H- and K-Ras in cells. The binding profile observed is typical of Ras-effectors, and SinRIP did not inhibit signalling by the Ras proteins, suggesting that it is not likely to be a Ras-inhibitor. It was also shown that SinRIP1 and SinRIP2 both interact and colocalise with c-Jun NH2- terminal kinase (JNK). Both SinRIP proteins were able to recruit JNK to their respective sub-cellular compartments. These interactions suggest an adaptor role for SinRIP in the Ras and/or JNK pathways. In addition, Sam68 was isolated as a SinRIP-binding protein in a yeast two-hybrid screen. Sam68 was shown to colocalise with SinRIP2 and endogenous SinRIP proteins, but not SinRIP1. Further colocalisation studies showed that endogenous SinRIP proteins localise in nuclear structures that may be associated with pre-mRNA splicing. Likely functions for SinRIP, as indicated by experimental results and studies of the orthologues of SinRIP in other species, are discussed.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Faculty of Science
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Thornton, Elizabeth Claire. "Identification and characterisation of a novel β subunit of AMP-activated protein kinase." Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312986.
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Schroder, Wayne Ashley, and n/a. "Cloning and Characterisation of the Human SinRIP Proteins." Griffith University. School of Biomolecular and Biomedical Science, 2003. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20030829.140754.
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This thesis describes the cloning and characterisation of a novel human gene and its protein products, which have been designated SAPK- and Ras-interacting protein (SinRIP). SinRIP shares identity with JC310, a partial human cDNA that was previously identified a candidate Ras-inhibitor (Colicelli et al., 1991, Proc Natl Acad Sci USA 88, p. 2913). In this study, it was shown that SinRIP is a member of an orthologous family of proteins that is conserved from yeast to mammals and contains proteins involved in Ras- and SAPK-mediated signalling pathways. Comparison of this family of proteins showed that human SinRIP contains a potential Ras-binding domain (RBD; residues 279-354), a PH-like domain (PHL; 376-487), and a highly conserved novel region designated the CRIM (134-265). Several other potential targeting sites, such as nuclear localisation signals and target sites for kinases, were identified within the SinRIP sequence. The human SinRIP gene is unusually large (>280 kbp) and is located on chromosome 9 at 9q34. SinRIP mRNA was detected in a wide variety of tissue-types and cell lines by RT-PCR, and the SinRIP sequences in the EST database were derived from an diverse array of tissues, suggesting a widespread or ubiquitous expression. Northern blot analysis revealed the highest levels in skeletal muscle and heart tissue. However, the steady-state levels of SinRIP mRNA vary greatly from cell to cell, and SinRIP expression is likely to be regulated at multiple post-transcriptional levels. It was shown that SinRIP mRNA is likely to be translated inefficiently by the normal cap-scanning mechanism, due to the presence of a GC-rich and structured 5-UTR, which also contains upstream ORFs. Alternative polyadenylation signals in the SinRIP 3-UTR can be used, resulting in the expression of short and long SinRIP mRNA isoforms. Several potential A/T-rich regulatory elements were also identified in SinRIP mRNA, which may target specific SinRIP mRNA isoforms for rapid degradation. Importantly, it was shown that SinRIP mRNA is alternatively spliced, resulting in the production of distinct SinRIP protein isoforms. Three isoforms, SinRIP2-4, were definitively identified by RT-PCR and full-length cloning. The SinRIP isoforms contain deletions in conserved regions, and are likely to have biochemical characteristics that are different to full-length SinRIP1. SinRIP2 is C-terminally truncated and lacks the PHL domain and part of the RBD, and relatively high levels of SinRIP2 expression arelikely to occur in kidneys. The RBD is disrupted in SinRIP3, but all other domains are intact, and RT-PCR analyses suggest that SinRIP3 is present in some cells at levels comparable to SinRIP1. A rabbit polyclonal antiserum against SinRIP was generated and detected endogenous SinRIP proteins. Using the anti-SinRIP antibody in immunoblots, multiple SinRIP isoforms were observed in most cell types. SinRIP1 and another endogenous SinRIP protein, likely to be SinRIP3, were detected in most cell lines, and appear to be are the major SinRIP proteins expressed in most cells. The subcellular localisation of both recombinant and endogenous SinRIP proteins was investigated by immunofluorescence assays and biochemical fractionation. Recombinant SinRIP1 protein was found in the cytoplasm and associated with the plasma membrane. In contrast, the SinRIP2 protein was predominantly nuclear, with only low-level cytoplasmic staining observed. The endogenous SinRIP proteins, likely to comprise these and other SinRIP isoforms, were found in both the nucleus and cytoplasm. SinRIP1 interacted with GTP-bound (active) Ras, but not GDP-bound (inactive) Ras, in an in vitro assay, and also co-localised with activated H- and K-Ras in cells. The binding profile observed is typical of Ras-effectors, and SinRIP did not inhibit signalling by the Ras proteins, suggesting that it is not likely to be a Ras-inhibitor. It was also shown that SinRIP1 and SinRIP2 both interact and colocalise with c-Jun NH2- terminal kinase (JNK). Both SinRIP proteins were able to recruit JNK to their respective sub-cellular compartments. These interactions suggest an adaptor role for SinRIP in the Ras and/or JNK pathways. In addition, Sam68 was isolated as a SinRIP-binding protein in a yeast two-hybrid screen. Sam68 was shown to colocalise with SinRIP2 and endogenous SinRIP proteins, but not SinRIP1. Further colocalisation studies showed that endogenous SinRIP proteins localise in nuclear structures that may be associated with pre-mRNA splicing. Likely functions for SinRIP, as indicated by experimental results and studies of the orthologues of SinRIP in other species, are discussed.
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Kanke, Toru. "Regulation of stress-activated protein kinases (SAPKs) mediated by proteinase-activated receptor-2 (PAR-2)." Thesis, University of Strathclyde, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.248779.
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Court, Naomi Wynne. "The subcellular localisation, tissue expression, substrate specificity and binding partners of stress-activated protein kinase-3." University of Western Australia. School of Biomedical and Chemical Sciences, 2004. http://theses.library.uwa.edu.au/adt-WU2004.0084.
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[Truncated abstract] Cells need to be able to detect changes in their surrounding environment and transduce these signals into the appropriate cellular compartments. One of the major ways that the cell achieves this signal transduction is through the process of phosphorylation. Protein kinases are the enzymes responsible for catalysing this transfer of phosphate groups from ATP to amino acid residues of their specific substrates. A subfamily of serine/threonine kinases known as the Mitogen-Activated Protein Kinases (MAPKs) is essential in a diverse range of cell processes including growth, metabolism, differentiation and death. The first identified MAPKs, the Extracellular Signal-Regulated Kinases (ERKs), were found to be activated in response to mitogenic stimuli such as growth factors. However, since the discovery of the ERKs, other pathways leading to the activation of related kinases have been recognised. These kinases are preferentially activated in response to stress, and are thus termed “Stress-Activated Protein Kinases” or SAPKs. They consist of the c-Jun N-terminal kinase isoforms 1, 2 and 3 (also called SAPK1γ, SAPK1α and SAPKβ respectively) and the p38 MAPKs, p38α, p38β, p38γ and p38δ (also called SAPK2a, SAPK2b, SAPK3 and SAPK4 respectively). A major challenge in this field has been to identify the substrates and functions of the SAPKs. This has been partly achieved by the development of inhibitors for the JNK MAPKs and SAPK2a/b. However, no inhibitors currently exist that specifically inhibit SAPK3 and SAPK4. Therefore, elucidating the function of these SAPKs has proved more difficult. Recent studies suggest that SAPK3 may play a unique role in the cell compared to other members of the p38 MAPK family. For example, several signalling proteins appear to specifically activate SAPK3 in certain circumstances while not activating other members of the p38 MAPK family. In addition, SAPK3 contains a unique sequence motif that allows it to bind to specialised domains known as PDZ domains. The interaction of SAPK3 with proteins containing these domains may regulate its subcellular localisation and interactions with other proteins in the cell. This project was undertaken to expand the knowledge on the expression, localisation, substrate specificity and binding partners of SAPK3. In Chapter 3 of this thesis, a SAPK3 monoclonal antibody was evaluated for its ability to specifically recognise endogenous SAPK3 protein. SAPK3 was found to be expressed in immortalised cell lines and primary cultures of neonatal rat myocytes, and to be colocalised with the mitochondria of these cells. This co-localisation remained unaltered in response to treatment with the nuclear export inhibitor Leptomycin B, and with exposure to osmotic shock, suggesting that SAPK3 substrates may be localised at the mitochondria
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Au-Yeung, Ka-wai. "Role of Chinese medicinal compounds in the regulation of stress-activated protein kinase in ischaemic/reperfused rat heart." Hong Kong : University of Hong Kong, 2000. http://sunzi.lib.hku.hk/hkuto/record.jsp?B22505441.
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Xing, Yu. "Mitogen activated protein kinase cascades mediate the regulation of antioxidant enzymes under abiotic stresses in arabidopsis." HKBU Institutional Repository, 2007. http://repository.hkbu.edu.hk/etd_ra/834.
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Goto, Ayumi. "Effects of acute heat stress on glucose metabolism and 5' adenosine monophosphate-activated protein kinase in skeletal muscle." Kyoto University, 2016. http://hdl.handle.net/2433/215632.
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Kyoto University (京都大学)0048
新制・課程博士
博士(人間・環境学)
甲第19806号
人博第777号
新制||人||187(附属図書館)
27||人博||777(吉田南総合図書館)
32842
京都大学大学院人間・環境学研究科共生人間学専攻
(主査)教授 林 達也, 教授 森谷 敏夫, 教授 石原 昭彦
学位規則第4条第1項該当
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Boppart, Marni D. "Regulation of stress-activated protein kinases by exercise and contraction in skeletal muscle." Thesis, Boston University, 2000. https://hdl.handle.net/2144/36769.
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Thesis (Sc.D.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
The c-Jun NH2-terminal kinase (JNK) and p38 intracellular signaling cascades are mitogen-activated protein kinase (MAPK) signaling pathways that are activated in mammalian cells by a variety of stressors, including proinflammatory cytokines, osmotic shock, and shear stress. The purpose of this dissertation research was to examine the effect of injury-producing exercise on JNK and p38 activities in human skeletal muscle and to determine whether mechanical stress is a primary stimulator of JNK and p38 activities with contraction. Twelve healthy subjects (7M/5F) completed maximal concentric or eccentric knee extensions on an isokinetic dynamometer (10 sets, 10 reps). Needle biopsies were obtained from the vastus lateralis muscle 24 h before exercise, immediately post-exercise, and 6 h post-exercise. While both forms of exercise increased JNK activity immediately post-exercise, eccentric contractions resulted in a much higher activation (15-fold vs. 4-fold increase above basal for eccentric and concentric, respectively). By 6 h post-exercise, JNK activity decreased back to baseline values. In a separate study, 14 male subjects completed a 42.2 km marathon. Biopsies were obtained from the vastus lateralis muscle 10 days prior to the marathon, immediately following the race, and 1, 3, and 5 days after the race. JNK activity increased 7-fold over basal immediately postexercise, but decreased back to basal 1, 3, and 5 days after the exercise. The activity of p38y also was increased and decreased in a similar pattern. However, no regulation was observed for p38α. In a third study, the effects of contraction and static stretch on JNK activity and p38 phosphorylation were determined in the rat soleus muscle in vitro. Static stretch dramatically increased JNK activity and p38 phosphorylation, whereas isometric contraction resulted in much smaller increases in JNK activity and p38 phosphorylation. The regulation of focal adhesion proteins also was examined following both exercise and contraction. The work presented in this thesis demonstrates that injury-producing exercise results in the marked activation of the JNK and p38 stress-activated protein kinases and provides evidence that mechanical stress may be a major contributor to increases in JNK and p38 activities observed following contraction in rat and human skeletal muscle.
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歐楊嘉慧 and Ka-wai Au-Yeung. "Role of Chinese medicinal compounds in the regulation of stress-activated protein kinase in ischaemic/reperfused rat heart." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2000. http://hub.hku.hk/bib/B31223916.
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Au-Yeung, Ka Wai, and 歐陽嘉慧. "A mechanistic study of the inhibitory effect of magnesium tanshinoate B on stress-activated protein kinase in ischaemia/reperfusion." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B2994188X.
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Pagano, Daniel J. (Daniel Joseph). "Genetic analysis of p38 mitogen-activated protein kinase signaling in innate immunity and stress physiology of Caenorhabditis elegans." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/106725.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2014.Cataloged from PDF version of thesis.
Includes bibliographical references.
Host-microbe interactions play an important role in the physiology and evolution of animals. Interactions with microbes can generally be considered beneficial or pathogenic to the host. The ability of an organism to mount an immune response to infection by pathogenic microbes is critical to its survival, and basic mechanisms of innate immunity are conserved in evolutionarily diverse species. A pivotal signaling pathway in the evolutionarily conserved innate immune responses of multicellular organisms is the stress-activated p38 mitogen-activated protein kinase (MAPK) pathway. This thesis focuses on the physiological role of p38 MAPK signaling in the host defense of Caenorhabditis elegans. In Chapter Two, I report the identification and characterization of the conserved ATF/CREB bZIP protein ATF-7 as the key transcriptional regulator of the PMK- 1 p38 MAPK-dependent immune response to pathogens. These data suggest a model in which the PMK- 1 p3 8 MAPK phosphorylates ATF-7 and switches it from a transcriptional repressor to a transcriptional activator of effector genes of the innate immune response of C. elegans. In Chapter Three, I characterize the roles of PMK- 1 and a second p38 MAPK ortholog, PMK-2, which are encoded in an operon, in tissue-specific signaling mechanisms involved in host defense. I show that PMK-2 functions redundantly with PMK- 1 in the nervous system to mediate neurobehavioral responses to pathogens. Furthermore, I demonstrate a role for the miR-58/80-82 family of microRNAs in regulating the tissue expression of pmk-2, which suggests a role for microRNAs in the establishment of tissue-specific expression of co-operonic genes. The work described in this thesis establishes the ancient evolutionary origins of the p38 MAPKCREB/ ATF pathway in innate immunity, and establishes a role for microRNAs in defining the tissue-expression pattern of co-operonic p38 MAPK genes. New directions for further understanding the ancient evolutionary mechanisms of p38 MAPK signaling and their tissue-specific regulation are discussed in Chapter Four.
by Daniel J. Pagano.
Ph. D.
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Lepez, Anouk. "Régulation du stress oxydant et contrôle de la prolifération homéostatique des lymphocytes T par l’AMP-activated protein kinase." Doctoral thesis, Universite Libre de Bruxelles, 2020. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/313133.
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Le nombre de lymphocytes T (LT) présents dans l’organisme est contrôlé de manière étroite et maintenu constant. En réponse à une chute de ce nombre, par exemple suite à une infection, un traitement de chimiothérapie ou lors d’une greffe de moelle osseuse, les LT résiduels ou transférés se divisent activement et repeuplent les compartiments lymphoïdes, rétablissant ainsi un nombre «normal» de cellules immunes périphériques. Ce processus, appelé prolifération homéostatique, est soutenu par l’IL-7 ainsi que les signaux TCR et mène à la génération de LT effecteurs/mémoire. Ces cellules effectrices/mémoire peuvent favoriser le développement d’une réponse anti-tumorale mais sont aussi impliquées dans le développement de maladies auto-immunes et inflammatoires.Des changements métaboliques sont étroitement liés à la différenciation et aux fonctions des LT. De plus, un nombre grandissant de données suggère qu’une modulation du métabolisme permet d’influencer le cours d’une réponse immune. L’AMP-activated protein kinase (AMPK) est un senseurmajeur du stress métabolique qui régule l’homéostasie des mitochondries, la glycolyse et l’équilibre rédox. Si l’AMPK est activée lors de la stimulation du TCR, son implication dans la biologie des LT reste confuse.Au cours de cette thèse, nous avons entrepris une série d’expériences afin d’évaluer le rôle de l’AMPK sur la biologie des LT et plus particulièrement au cours de leur prolifération homéostatique. Grâce à différents modèles in vivo et in vitro, nous avons montré que l’AMPK, bien que dispensable pour les étapes précoces de l’activation du TCR, est requise pour soutenir la viabilité et l’expansion des LT au cours de proliférations de longues durées. L’AMPK promeut l’accumulation de LT effecteurs/ mémoire au cours d’une prolifération homéostatique. En accord avec ces données, la transplantation de LT AMPK-KO dans un hôte allogénique conduit au développement d’une réaction du greffon contre l’hôte de moindre gravité.D’un point de vue métabolique, l’AMPK soutient le potentiel de membrane mitochondrial, permet une plus grande flexibilité métabolique, limite la production de dérivés toxiques de l’oxygène (ROS) et protège les LT contre le stress oxydant. En outre, la neutralisation des ROS par un traitement antioxydant restaure partiellement la prolifération des LT AMPK-KO.Nos données suggèrent qu’en limitant l’accumulation de dégâts mitochondriaux et oxydatifs au cours de cycles de divisions prolongées, l’AMPK soutient la viabilité, la prolifération et les fonctions effectrices des LT.Doctorat en Sciences
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Shahidullah, Mohammad, Amritlal Mandal, and Nicholas A. Delamere. "A Role for Calcium-Activated Adenylate Cyclase and Protein Kinase A in the Lens Src Family Kinase and Na,K-ATPase Response to Hyposmotic Stress." ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2017. http://hdl.handle.net/10150/625814.
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PURPOSE. Na, K-ATPase activity in lens epithelium is subject to control by Src family tyrosine kinases (SFKs). Previously we showed hyposmotic solution causes an SFK-dependent increase in Na, K-ATPase activity in the epithelium. Here we explored the role of cAMP in the signaling mechanism responsible for the SFK and Na, K-ATPase response. METHODS. Intact porcine lenses were exposed to hyposmotic Krebs solution (200 mOsm) then the epithelium was assayed for cAMP, SFK phosphorylation (activation) or Na, K-ATPase activity. RESULTS. An increase of cAMP was observed in the epithelium of lenses exposed to hyposmotic solution. In lenses exposed to hyposmotic solution SFK phosphorylation in the epithelium approximately doubled as did Na, K-ATPase activity and both responses were prevented by H89, a protein kinase A inhibitor. The magnitude of the SFK response to hyposmotic solution was reduced by a TRPV4 antagonist HC067047 added to prevent TRPV4-mediated calcium entry, and by a cytoplasmic Ca2+ chelator BAPTA-AM. The Na, K-ATPase activity response in the epithelium of lenses exposed to hyposmotic solution was abolished by BAPTA-AM. As a direct test of cAMP-dependent SFK activation, intact lenses were exposed to 8-pCPT-cAMP, a cell-permeable cAMP analog. 8-pCPT-cAMP caused robust SFK activation. Using Western blot, two calcium-activated adenylyl cyclases, ADCY3 and ADCY8, were detected in lens epithelium. CONCLUSIONS. Calcium-activated adenylyl cyclases are expressed in the lens epithelium and SFK activation is linked to a rise of cAMP that occurs upon hyposmotic challenge. The findings point to cAMP as a link between TRPV4 channel-mediated calcium entry, SFK activation, and a subsequent increase of Na, K-ATPase activity.
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Haberstroh, Katharine Michelle Wendy. "Cadmium-induced cell toxicity and stress-activated protein kinase induction in the midbrain cells of two strains of mice." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp03/MQ40474.pdf.
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Chang, Malcolm Elliott. "Role of the stress activated protein kinases (sapk's) in mediating resistance to the antineoplastic agent adriamycin (ADR)." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape11/PQDD_0014/MQ40835.pdf.
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Combes, Adrien. "Influence des perturbations métaboliques sur des voies de signalisation impliquées dans la biogenèse mitochondriale." Thesis, Lille 2, 2015. http://www.theses.fr/2015LIL2S045/document.
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L’évolution des populations occidentales s’accompagne d’une augmentation de la sédentarité et des maladies métaboliques qui accroissent les problèmes de santé. Ces évolutions ont des répercussions sur le muscle squelettique qui voit sa capacité à produire de l’énergie aérobie diminuer. Néanmoins, le muscle squelettique est très plastique et les capacités oxydatives musculaires s’améliorent rapidement par l'activité physique. Les mitochondries sont des éléments majeurs des capacités oxydatives musculaires et la compréhension des mécanismes moléculaires qui régissent la biogenèse et la fonction mitochondriale est nécessaire pour prescrire au mieux l’activité physique.L’exercice intermittent semble être de plus en plus utilisé dans la pratique. Plusieurs arguments sont mis en avant pour préconiser cette modalité : 1) le temps passé à haute consommation d’oxygène, 2) la haute intensité et 3) les perturbations métaboliques induites par les variations d’intensité au cours de l’exercice. Cependant, l’influence des perturbations métaboliques sur les capacités oxydatives musculaires n’a pas encore été clairement démontrée. L’objet des mes travaux de thèse s’est donc focalisé sur ces perturbations métaboliques et leurs effets sur les voies de signalisation impliquées dans la biogenèse mitochondriale. Afin de caractériser l’implication des perturbations métaboliques dans la stimulation des voies de signalisation de la biogenèse mitochondriale, nous avons comparé l’influence d’exercices aigus sur ces voies de signalisation. Deux protocoles nous ont permis d’investiguer l’influence des variations métaboliques. Le premier a consisté, lors d’un exercice de intermittent, à identifier la durée du cycle induisant les plus grandes perturbations métaboliques et à caractériser les effets de la modalité d’exercice sur un exercice de 30 minutes de pédalage à 70%WRpic. Le second protocole visait à déterminer l’influence de la répétition des perturbations métaboliques sur les voies de signalisation régulant la biogenèse mitochondriale.Afin d’identifier la durée de cycle produisant le plus de variations métaboliques, nous avons analysé l’évolution de la consommation d’oxygène et quantifié les variations métaboliques. Pour cela nous avons utilisé trois paramètres : 1) un paramètre quantitatif, 2) un paramètre qualitatif et 3) un index associant les paramètres quantitatif et qualitatif. La comparaison de trois durées de cycle différentes (30s d’effort:30s de récupération passive ; 60s:60s et 120s:120s) nous a permis de mettre en évidence que la modalité 60s:60s est celle qui induit le plus de variations métaboliques et cela pour une dépense énergétique identique pour les trois modalités.Notre seconde étude a consisté à comparer 30 minutes de pédalage à 70%WRpic sous deux modalités différentes : continue (1 bloc de 30min) et intermittente (30 bloc de 1min entrecoupés de 1min). La répétition de phase d’exercice et de repos lors de l’exercice intermittent créée plus de perturbation du métabolisme et entraîne une phosphorylation supérieure de l'AMPK, CaMKII et p38 MAPK. Ces kinases sont situées en amont de PGC-1α, un important régulateur de la biogenèse mitochondriale dans le muscle squelettique. Ces résultats mettent donc en évidence un effet spécifique des perturbations métaboliques sur les voies de signalisation contrôlant la biogenèse mitochondriale.Ces travaux ouvrent de nouvelles perspectives sur les méthodes de réentraînement de personnes sédentaires ou atteintes de pathologie chronique. Les futurs travaux viseront à confirmer nos résultats lors d’interventions chroniques et d’explorer ces effets chez différentes populationsWestern life evolution is associated with an increase in sedentary behaviours and metabolic diseases leading to health alteration. This evolution affects the skeletal muscle, which is characterized by a decrease in its ability to produce aerobic energy. However, skeletal muscle is a highly malleable tissue, capable of considerable metabolic adaptations in response to physical activity. Mitochondria produce the aerobic energy within the skeletal muscle. Understanding the molecular mechanisms that regulate mitochondrial biogenesis and its function is necessary to improve physical activity prescription.The intermittent exercise is currently used in rehabilitation programs. Several arguments are put forward to utilizing this method: 1) the time spent at high oxygen consumption, 2) the high intensity of exercise and 3) the metabolic disturbances induced by variations of intensity during exercise. However, the influence of metabolic disturbances on muscle oxidative capacity has not been clearly demonstrated. The purpose of my thesis work has therefore focused on these metabolic perturbations and their effects on signalling pathways involved in mitochondrial biogenesis. In order to characterize the influence of metabolic disturbances on the signalling pathways involved in mitochondrial biogenesis, we compared the influence of acute exercises. We realized two protocols to investigate the influence of metabolic disturbances. The first study compared three intermittent exercises in order to identify the optimal duty-cycle duration to induce the biggest metabolic disturbances and to compare metabolic responses of intermittent and continuous exercise performed at 70%WRpic. The second protocol evaluated the influence of the repetition of metabolic disturbances on signalling pathways involved in mitochondrial biogenesis.In order to identify the duty-cycle duration producing more metabolic fluctuations, we analysed the changes of oxygen consumption and quantified metabolic variations. We used three parameters: 1) a quantitative parameter, 2) a qualitative parameter, and 3) an index combining quantitative and qualitative parameters. Comparison of three different duty-cycle durations (30s work:30s passive recovery; 60s:60s, and 120s:120s) revealed that the 60s:60s modality induces more metabolic fluctuations for a same energy expenditure.Our second study compared 30 minutes of pedalling at 70%WRpic realized by two different modalities: continuous (30min 1 block) and intermittent (30 1min block interspersed by 1min of passive recovery). Repetition of transitions from rest to exercise during the intermittent exercise creates higher metabolic disturbances and leads to a higher phosphorylation of AMPK, p38 MAPK and CaMKII. These kinases are upstream of PGC-1α, an important regulator of mitochondrial biogenesis in skeletal muscle. All together, these results demonstrate that metabolic disturbances are involved in mitochondrial signalling pathways activation.This work opens up new perspectives on exercise training prescription for sedentary or chronic pathology people. Future work will aim to confirm our results in chronic interventions and explore these effects in different populations
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Launay, Nathalie. "Voies de signalisation et rôle de l'αB-cristalline suite à la désorganisation du cytosquelette." Paris 6, 2006. http://www.theses.fr/2006PA066375.
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Le premier objectif de ce travail de recherche a été d’identifier les voies de signalisation impliquées dans la transduction du signal engendré par la perturbation/désorganisation spécifique des trois principales structures du cytosquelette au sein du système musculaire. Ces stress spécifiques se caractérisent par l’induction de la phosphorylation spécifique de l’B-cristalline, modification qui induit des changements de localisation et régulerait les fonctions de la protéine en augmentant son affinité pour certains éléments du cytosquelette. Dans un deuxième temps, le rôle de la phosphorylation de l’B-cristalline a été étudié dans le phénomène de résistance de cellules de carcinomes mammaires vis-à-vis des traitements anti-tumoraux altérant le cytosquelette. Lors de ce travail, nous avons mis en évidence que la phosphorylation du résidu sérine 59 de l’B-cristalline jouerait un rôle clé dans la régulation des propriétés anti-apoptotiques de la protéineThe first aim of this research was to identify the pathways involved in the signal transduction induced by specific cytoskeletal stresses in the muscular system. Cytoskeletal network disruption trigger the specific B-crystallin phosphorylation which may protect or stabilize the actin microfilaments and interfere with formation of IF aggregates during stresses exerted at the cytoskeleton. The second aim of this project was to study the role of phosphorylation in the regulation of the αB-crystallin anti-apoptotic function in chemotherapy resistance phenomenon in breast epithelial adenocarcinoma cells. We demonstrate that Ser59 phosphorylation of B-crystallin plays a crucial role in vinblastine-induced apoptosis in MCF7 cells, by down-regulating the anti-apoptotic function of B-crystallin. These results introduce the possibility of regulating the protective status of sHsp in cancer through modifying their state of phosphorylation
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Sama, Reddy Ranjith Kumar. "FUS/TLS in Stress Response - Implications for Amyotrophic Lateral Sclerosis: A Dissertation." eScholarship@UMMS, 2014. http://escholarship.umassmed.edu/gsbs_diss/704.
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Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease is a fatal neurodegenerative disease. ALS is typically adult onset and is characterized by rapidly progressive loss of both upper and lower motor neurons that leads to death usually within 3-5 years. About 90% of all the cases are sporadic with no family history while the remaining 10% are familial cases with mutations in several genes including SOD1, FUS/TLS, TDP43 and C9ORF72.
FUS/TLS (Fused in Sarcoma/Translocated in Liposarcoma or FUS) is an RNA/DNA binding protein that is involved in multiple cellular functions including DNA damage repair, transcription, mRNA splicing, RNA transport and stress response. More than 40 mutations have now been identified in FUS that account for about 5% of all the familial cases of ALS. However, the exact mechanism by which FUS causes ALS is unknown. While significant progress has been made in understanding the disease mechanism and identifying therapeutic strategies, several questions still remain largely unknown. The work presented here aims at understanding the normal functions of FUS as well as the pathogenic mechanisms by which it leads to disease.
Several studies showed the association of mutant-FUS with structures made up of RNA and proteins, called stress granules that form under various stress conditions. However, little is known about the role of endogenous FUS under stress conditions. I have shown that under hyperosmolar conditions, the predominantly nuclear FUS translocates into the cytoplasm and incorporates into stress granules. The response is specific to hyperosmolar stress because FUS remains nuclear under other stress conditions tested, such as oxidative stress, ER stress and heat shock. The response of FUS is rapid, and cells with reduced FUS levels are susceptible to the hyperosmolar stress, indicating a pro-survival role for FUS. In addition to investigating the functions of endogenous wild-type (WT) FUS, the work presented also focuses on identifying the pathogenic mechanism(s) of FUS variants. Using various biochemical techniques, I have shown that ALS-causing FUS variants are misfolded compared to the WT protein. Furthermore, in a squid axoplasm based vesicle motility assay, the FUS variants inhibit fast axonal transport (FAT) in a p38 MAPK dependent manner, indicating a role for the kinase in mutant-FUS mediated disease pathogenesis. Analysis of human ALS patient samples indicates higher levels of total and phospho p38, supporting the notion that aberrant regulation of p38 MAPK is involved in ALS.
The results presented in this dissertation 1) support a novel prosurvival role for FUS under hyperosmolar stress conditions and, 2) demonstrate that protein misfolding and aberrant kinase activation contribute to ALS pathogenesis by FUS variants.
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Lu, Dan. "ATF3, a stress-inducible gene function and regulation /." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1155740569.
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Guillonneau, Maëva. "Étude de la nucléophosmine, nouvelle protéine associée à p38 MAPK, dans la réponse des cellules endothéliales à un stress oxydant aigu." Nantes, 2015. https://archive.bu.univ-nantes.fr/pollux/show/show?id=288675a2-942d-436d-b15a-59ae37d33154.
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Le compartiment microvasculaire est une cible importante du stress oxydant qui est un facteur majeur de la dysfonction endothéliale, notamment au cours d'exposition aux rayonnements ionisants. L'altération de l'endothélium induite par le stress oxydant est impliquée dans la toxicité radio-induite des tissus sains. Limiter les dysfonctions endothéliales est donc un enjeu important des traitements radiothérapeutiques actuels. Cet objectif nécessite une meilleure caractérisation de la signalisation du stress oxydant dans les cellules endothéliales. La voie p38 MAPK est incontournable dans la réponse au stress oxydant mais reste encore insuffisamment caractérisée. Par une approche protéomique, nous avons identifié la nucléophosmine (NPM) comme nouveau partenaire de p38 dans le cytoplasme des cellules endothéliales. La phosphatase PP2a est aussi associée à ce complexe NPM/p38. Nos travaux montrent que le stress oxydant (H2O2, 500μM) régule la déphosphorylation de NPM via PP2a, entraine sa dissociation rapide du complexe et favorise sa translocation vers le noyau. De plus, nous montrons que la présence de NPM déphosphorylée au noyau altère la réponse des cellules aux dommages à l'ADN induits par le stress oxydant. Le céramide sphingolipide membranaire est également un facteur important des voies de stress, particulièrement dans les cellules endothéliales. Notre étude aborde donc l'implication de ce sphingolipide dans la régulation de la voie NPM/p38. Une meilleure caractérisation de la voie p38 et de ses acteurs permettra d'identifier de potentielles cibles afin de limiter les dysfonctions endothéliales et leurs conséquences délétères sur les tissus environnantsThe microvascular compartment is a significant target of oxidative stress that is a major factor in endothelial dysfunction, especially during exposure to ionizing radiation. The alteration of endothelium induced by oxidative stress is involved in radiation-induced toxicity of normal tissues. Limiting endothelial dysfunction is therefore an important issue of current radiotherapeutic treatments. This objective requires a better characterization of oxidative stress signaling in endothelial cells. P38 MAPK pathway is essential in oxidative stress response but still insufficiently characterized. By using a proteomic approach, we identified nucleophosmin (NPM) as a new partner of p38 in the cytoplasm of endothelial cells. PP2a phosphatase is also associated with the NPM/p38 complex. Our work shows that oxidative stress (H2O2, 500μM) regulates the NPM dephosphorylation via PP2a, causes rapid dissociation of the complex, and promotes its translocation to the nucleus. In addition, we show that the presence of NPM dephosphorylated at T199 in the nucleus alters the cellular response to DNA damage induced by oxidative stress. The membrane sphingolipid ceramide is also an important factor in stress pathways, particularly in endothelial cells. Our study describes the involvement of this sphingolipid in the regulation of NPM/p38 pathway. A better characterization of the p38 pathway and its actors provided by our study will identify potential targets in order to limit endothelial dysfunction and its deleterious effect on surrounding tissues
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Schaeffer, Céline. "Effets protecteurs d'un neuropeptide, le CGRP, sur des cellules du système cardiovasculaire soumises à un stress oxydant." Dijon, 2003. http://www.theses.fr/2003DIJOMU14.
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Des cellules musculaires lisses de rat ont été soumises à un stress radicalaire induit par un système générateur de peroxyde d'hydrogène : le système Glucose-Glucose Oxydase ce qui entraîne la mort cellulaire par apoptose. Un prétraitement des cellules avec du h-CGRP diminue significativement ce propessus d'apoptose. L'étude de l'influence du prétraitement par le CGRP a également été réalisée sur des cultures de cardiomyocytes de rat nouveau né soumis à une ischémie/reperfusion simulée in vitro. Le prétraitement par le CGRP réduit significativement la perte de cellules à la reperfusion et améliore le rétablissement fonctionnel lors de la reperfusion. L'action cytoprotectrice exercée sur les CML est due à une suractivation des MAP kinases ERK 1/2 et P38. Ces sur-activations sont provoquées par la fixation du CGRP sur le récepteur spécifique de la famille CGRP/adrénomédulline. De plus, la PKC ainsi que la protéine Ki-Ras sont également activées par cette fixation du CGRP au récepteur. Le CGRP agit également sur la production de radicaux libres puisque leur présence est diminuée lorsque les cellules sont prétraitées par le CGRP.
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Cameron, Pamela. "The cytotoxic and inflammatory effects of E. coli 0157:H7 : the role of stress-activated protein kinases and nuclear factor kappa B." Thesis, University of Strathclyde, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366792.
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Bertran-Gonzalez, Jesus. "Study of segregated signaling responses of striatonigral and striatopallidal neurons in BAC transgenic mice." Paris 6, 2009. http://www.theses.fr/2009PA066348.
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Le striatum reçoit et intègre des informations arrivant de régions corticales et thalamiques et de neurones dopaminergiques du mésencéphale, formant ainsi la station d'entrée principale au circuit des ganglions de la base (GB). Il est principalement composé de deux sous-types de neurones GABAergiques épineux de taille moyenne (MSNs), qui projettent vers différents noyaux. La voie directe est constituée par les neurones striatonigraux, qui projettent directement vers la substance noire pars reticulé (SNr), la station de sortie des GB. La voie indirecte lie le striatum et la SNr par des relais intermédiaires, dont les premières projections sont ceux des neurones striatopallidaux. Etant donné leurs influences opposées au niveau de la SNr, les informations transmises par les neurones striatonigraux et striatopallidaux à travers des circuits thalamocorticaux sont cruciales pour la sortie finale des GB. La dopamine (DA) module les informations corticostriatales et thalamostriatales au travers des différents types de récepteurs de la DA exprimés dans les MSNs. Les récepteurs de type D1 (D1R) sont connus pour activer des programmes importants de la signalisation moléculaire tels que les modules cAMP/PKA/DARPP-32 et MAPK/ERK, tous deux usant de réponses cytoplasmiques et nucléaires essentielles. D'autre part, les récepteurs de type D2 (D2R) sont négativement associés à la formation de l’AMPc, et sont fonctionnellement couplés aux récepteurs d'adénosine A2A (A2AR) dans un jeu opposé qui régule des réactions intracellulaires et au niveau de la membrane. À la lumière de la controverse existant encore sur la distribution ségrégée vs superposée des sous-types de récepteurs de la DA dans les neurones striatonigraux et striatopallidaux, nous avons entrepris une série d'études neuro-anatomiques et histologiques pour la caractérisation des souris transgéniques BAC drd1a-EGFP et drd2-EGFP, qui marquent les cellules exprimant D1R et D2R, respectivement. Nous avons constaté que les D1R et D2R sont respectivement distribués dans les neurones striatonigraux et striatopallidaux de manière très ségrégée, et nous avons estimé les proportions de neurones exprimant D1R, D2R ou les deux dans différentes régions du striatum. Nos résultats appuient l'organisation des GB dans les voies directe et indirecte, mais nous avons détecté la présence de certains terminaux des D1R-MSNs au niveau du globus pallidus latéral. Surtout, nous avons clairement démontré que, après des traitements de cocaïne aigus et chroniques, à la fois des réponses cytoplasmiques -révélées par l'activation de ERK- et réponses nucléaires -révélées par l'activation de MSK1 et la phosphorylation de l'histone H3- se produisent exclusivement dans les neurones striatonigraux, une distribution qui est largement suivie plus tard par l'induction des gènes immédiats précoces c-fos et zif268. Nous démontrons également que l’antipsychotique typique halopéridol, ainsi que des inhibiteurs spécifiques des D2R, activent toutes ces réponses de signalisation sélectivement dans les neurones striatopallidaux, par un mécanisme de dérépression des A2AR. En parallèle, nous révélons des régulations moléculaires exclusives des réponses nucléosomales dans ces cellules. Dans l'ensemble, les résultats présentés dans cette thèse mettent en évidence une forte ségrégation histologique et fonctionnelle des neurones du striatum, ce qui est essentiel pour la compréhension de la modulation des GB exercée par la DA dans le striatum.
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De, Silva Matharage Shenali. "Involvement of AMPK and AP-1 Biochemical Pathways in IL-6 Regulation of Steroidogenic Enzymes in the Adrenal Cortex." BYU ScholarsArchive, 2013. https://scholarsarchive.byu.edu/etd/4301.
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The adrenal cortex is a crucial endocrine gland in the mammalian stress response. In chronic inflammatory stress, cortisol is elevated whereas adrenal androgens are decreased. Furthermore, ACTH levels have poor correlation with the plasma cortisol in these conditions, thus suggesting that other factors are driving the stress response during chronic inflammatory stress. Interleukin-6 (IL-6), a cytokine which is released during chronic inflammatory stress, is assumed to be one such factor. Thus the biochemical pathways by which IL-6 increases cortisol release from the zona fasciculata (ZF), and decreases adrenal androgen release from the zona reticularis (ZR) were investigated. Since IL-6 activates AMP-activated kinase (AMPK) in skeletal muscle, AMPK was investigated for IL-6- induced effects in ZF and ZR tissue. The effects of AMPK activation and IL-6 exposure on the expression of the steroidogenic proteins, steroidogenic acute regulatory protein (StAR) and cholesterol side chain cleavage enzyme (P450scc), and on the steroidogenic nuclear factors steroidogenic factor-1 (SF-1) and adrenal hypoplasia congenita, critical region on the X chromosome, gene-1 (DAX-1) were investigated. AMPK activation and IL-6 exposure increased the expression of StAR, P450scc, and SF-1, and decreased DAX-1 in the ZF. Meanwhile, AMPK activation and IL-6 exposure decreased the expression of StAR, P450scc, and SF-1, and increased DAX-1 in the ZR. AMPK inhibition blocked the effects of AMPK activation and IL-6 on the ZF and ZR. Activator Protein-1 (AP-1) was the second biochemical intermediate studied since in other tissues AMPK activation increases the expression and phosphorylation of AP-1 subunits. IL-6 stimulation and AMPK activation increased the expression of the AP-1 subunits cFOS, cJUN, JUN B, and JUN D, while increasing the phosphorylation of cJUN in both the ZF and the ZR. These effects were blocked by AMPK inhibition. Inhibition of AP-1 leads to decreased StAR, P450scc, and SF-1, and increased DAX-1 in the ZF. Meanwhile, AP-1 inhibition leads to increased StAR, P450scc, SF-1, and decreased DAX-1 in the ZR. Therefore the AP-1 complex functions as a biochemical intermediate in the IL-6 and AMPK regulation of steroidogenic enzymes in the ZF and ZR. Overall, the results suggest that IL-6 activates AMPK, which increases the expression and phosphorylation of AP-1 subunits in the ZF and the ZR. However, increased AP-1 activation leads to increased StAR and P450scc in the ZF, but decreased StAR and P450scc in the ZR.
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Jacob, Claire. "Régulation du stress oxydant par les phosphodiestérases de type 4 : implication des MAP kinases." Rennes 1, 2003. http://www.theses.fr/2003REN1B069.
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Nous avons montré : 1/ dans les neutrophiles de rat que les phosphodiestérases de type 4 (PDE4) inhibaient ERK1/2 par une interaction directe et que les inhibiteurs PDE4 inhibaient le production d'O2[Anion superoxyde] induite par le fMLP par une activation de ERK1/2 indépendante de l'AMPc. 2/ chez le patient atteint de bronchopneumopathie chronique obstructive (BPCO) que les inhibiteurs PDE4 étaient plus efficaces dans les cellules mononuclées que dans les neutrophiles pour inhiber la production d'O2 induite par le fMLP et que cette inhibition se faisait par une voie dépendante de l'AMPc et par une inhibition directe de ERK1/2. Cette étude nous a permis d'en déduire une application au traitement des BCPO : l'association d'un inhibiteur PDE4 avec un activateur de PKA ou avec un inhibiteur de MEK1/2 pourrait augmenter l'efficacité du traitement sur la production d'O2 qui entretient l'état inflammatoire du tissu pulmonaire.
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Noriega, Esteban Núria. "The Rtg1 and Rtg3 proteins are novel transcription factors regulated by the yeast hog1 mapk upon osmotic stress." Doctoral thesis, Universitat Pompeu Fabra, 2009. http://hdl.handle.net/10803/7158.
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La adaptación de la levadura Saccharomyces cerevisiae a condiciones de alta osmolaridad está mediada por la vía de HOG ((high-osmolarity glycerol). La activación de esta vía induce una serie de respuestas que van a permitir la supervivencia celular en respuesta a estrés. La regulación génica constituye una respuesta clave para dicha supervivencia. Se han descrito cinco factores de transcripción regulados por Hog1 en respuesta a estrés osmótico. Sin embargo, éstos no pueden explicar la totalidad de los genes regulados por la MAPK Hog1. En el presente trabajo describimos cómo el complejo transcripcional formado por las proteínas Rtg1 y Rtg3 regula, a través de la quinasa Hog1, la expresión de un conjunto específico de genes. Hog1 fosforila Rtg1 y Rtg3, aunque ninguna de estas fosforilaciones son esenciales para regulación transcripcional en respuesta a estrés. Este trabajo también muestra cómo la deleción de proteínas RTG provoca osmosensibilidad celular, lo que indica que la integridad de la vía de RTG es esencial para la supervivencia celular frente a un estrés osmótico.In Saccharomyces cerevisiae the adaptation to high osmolarity is mediated by the HOG (high-osmolarity glycerol) pathway, which elicits different cellular responses required for cell survival upon osmostress. Regulation of gene expression is a major adaptative response required for cell survival in response to osmotic stress. At least five transcription factors have been reported to be controlled by the Hog1 MAPK. However, they cannot account for the regulation of all of the genes under the control of the Hog1 MAPK. Here we show that the Rtg1/3 transcriptional complex regulates the expression of specific genes upon osmostress in a Hog1-dependent manner. Hog1 phosphorylates both Rtg1 and Rtg3 proteins. However, none of these phosphorylations are essential for the transcriptional regulation upon osmostress. Here we also show that the deletion of RTG proteins leads to osmosensitivity at high osmolarity, suggesting that the RTG-pathway integrity is essential for cell survival upon stress.
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Streicher, John Michael. "The role of mitogen activated protein kinase activated protein kinase-2 in regulating p38 mitogen activated protein kinase induced cyclooxygenase-2 induction and heart failure." Diss., Restricted to subscribing institutions, 2009. http://proquest.umi.com/pqdweb?did=1872200951&sid=6&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Underwood, E. A. "Nucleotide regulation of AMP-activated protein kinase." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1382013/.
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AMP-activated protein kinase (AMPK) acts as the cell’s master energy regulator, sensing and maintaining the concentration of ATP in a narrow range irrespective of energy demand. This kinase has received significant attention as a drug-target for type-2 diabetes, obesity and cancer. Although historically the AMP:ATP ratio has been considered the signal for AMPK activation, we have recently demonstrated that ADP is likely to be an important physiological regulator of AMPK in both mammals and yeast. The binding of adenine nucleotides and staurosporine to the full-length alpha1beta1gamma1 heterotrimer, both phosphorylated and unphosphorylated, is described. Binding was monitored through displacement of fluorescently labelled nucleotides (coumarin-AXP), either via direct coumarin excitation or Forster Resonance Energy Transfer (FRET) in which tryptophan residues were excited. Mg.ATP was found to bind more weakly than ADP, a feature which is likely key to AMPK regulation. A Nicotinamide adenine dinucleotide (NADH) coupled spectrophotometric assay was used to monitor AMPK kinetics and its regulation by nucleotides. NADH binds at Site-1, within the gamma-subunit, and competes with allosteric activation by AMP, but not the protective effect of AMP/ADP against alpha-T172 dephosphorylation. Therefore it seems that AMP binding at Site-1 mediates allostery whilst AMP/ADP binding at Site-3 affords protection against dephosphorylation. In order to explore this idea further, AXP binding constants were used to model binding site occupancies over the concentration ranges used in vitro. The modelling demonstrates that, in vitro, Site-1 is occupied by AMP and Site-3 by AMP/ADP in a manner consistent with their assigned regulatory functions. This modelling study was also extended to consider in vivo binding site occupancy. It was important to verify that coumarin-ADP bound in a homologous fashion to ADP, specifically in the same exchangeable binding pockets. X-ray crystallography was used to determine the structure of a truncated form of AMPK in complex with coumarin-ADP. This structure is compared to an ADP-bound form. SNF1 is the Saccharomyces cerevisiae AMPK ortholog. The binding of nucleotides to SNF1, and its regulation by ADP, was also characterised. As with the mammalian enzyme AXP bound at two exchangeable sites, and interacted with Mg.ATP more weakly than ADP.
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Davies, Gareth. "Folding of p38 mitogen-activated protein kinase." Thesis, University of Warwick, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.412847.
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Scheffler, Tracy Leigh. "AMP-activated protein kinase and muscle metabolism." Diss., Virginia Tech, 2012. http://hdl.handle.net/10919/38829.
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AMP-activated protein kinase (AMPK) is a major regulator of skeletal muscle metabolism with relevance to agriculture and human health. During the conversion of muscle to meat, the rate and extent of postmortem metabolism and pH decline largely determine pork quality development. Pigs with the AMPKγ3 R200Q mutation generate pork with low ultimate pH (pHu); this is attributed to high glycogen content, and greater â potentialâ to produce lactate and H+. We hypothesized that decreasing muscle phosphocreatine and creatine would decrease ATP buffering capacity, resulting in earlier termination of glycolysis and pH decline. Dietary supplementation with the creatine analogue, β-GPA, decreased muscle total creatine but negatively affected performance. Another experiment was conducted using control or β-GPA diet and wild type and AMPKγ3R200Q pigs in a 2à 2 factorial design. The loss of muscle total creatine was important in maintenance of ATP levels in AMPKγ3R200Q muscle early postmortem. Moreover, elevated glycogen did not affect pHu, supporting that energetic modifications induced by feed restriction and β-GPA supplementation influence extent of pH decline. Next, we utilized a line of pigs selected for differences in pHu. Another AMPKγ3 mutation (V199I), which is associated with higher pHu and lower glycolytic potential, was prevalent. The 199II genotype increased pHu in castrated males only. The wild type VV genotype increased glycolytic potential, but neither glycolytic potential nor lactate predicted pHu.
In humans, AMPK activation is at least partly responsible for the beneficial effects of exercise on glucose transport and increased oxidative capacity in skeletal muscle. An inverse relationship exists between skeletal muscle fiber cross-sectional area and oxidative capacity, which suggests muscle fibers hypertrophy at the expense of oxidative capacity. Therefore, we utilized pigs possessing mutations associated with increased oxidative capacity (AMP-activated protein kinase, AMPKγ3R200Q) or fiber hypertrophy (ryanodine receptor 1, RyR1R615C) to determine if these events occur in parallel. RyR1R615C increased muscle fiber size; AMPKγ3R200Q increased oxidative capacity, evidenced by enhanced enzyme activity, mitochondrial function, and expression of mitochondrial proteins. Thus, pigs with both AMPKγ3R200Q and RyR1R615C possess increased fiber size and oxidative capacity, suggesting hypertrophy and oxidative capacity can occur simultaneously in skeletal muscle.Ph. D.
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Hurst, Denise. "AMP-activated protein kinase kinase activity and phosphorylation of AMP-activated protein kinase in contracting muscle of sedentary and endurance trained rats." Diss., CLICK HERE for online access, 2007. http://contentdm.lib.byu.edu/ETD/image/etd2014.pdf.
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Brown, Jacob D. "Liver Kinase B1/AMP-Activated Protein Kinase Signaling in the Diaphragm." BYU ScholarsArchive, 2010. https://scholarsarchive.byu.edu/etd/2543.
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The Liver Kinase B1 (LKB1)/AMP-Activated Protein Kinase (AMPK) signaling pathway is a major regulator of skeletal muscle metabolic processes. During exercise, LKB1-mediated phosphorylation of AMPK leads to its activation, promoting mitochondrial biogenesis and glucose transport, among other effects. The roles of LKB1 and AMPK have not been fully characterized in the diaphragm. Two methods of AMPK activation were used to characterize LKB1/AMPK signaling in diaphragms from muscle-specific LKB1 knockout (KO) and littermate control (C) mice: (1) acute injection of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and (2) 5-min direct electrical stimulation (ES) of the diaphragm. Diaphragms were excised 60 minutes post-AICAR injection and immediately after ES. AMPK phosphorylation increased with AICAR and ES in C but not KO mice. Acetyl CoA carboxylase (ACC) phosphorylation increased with AICAR in C but not KO mice, but increased in both genotypes with ES. While the majority of mitochondrial enzyme levels were lower in KO diaphragms, uncoupling protein 3 (UCP-3) levels were not different between genotypes. A IIx to IIb fiber type switch was observed in KO diaphragms. While in vitro peak force generation was similar between genotypes, KO diaphragms fatigued more quickly and had an impaired ability to recover. In conclusion, LKB1 regulates AMPK phosphorylation, mitochondrial enzyme expression, fiber type distribution, as well as recovery of the diaphragm from fatigue.
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Svensson, Nilsson Caroline. "Post-translational modification of protein kinase RNA-activated." Thesis, Uppsala universitet, Institutionen för kemi - BMC, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-355076.
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Rojnuckarin, Ponlapat. "Mitogen-activated protein kinase pathways in megakaryocyte development /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/9200.
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Julien, Mathéau A. "Mechanical Strain-Mediated Syndecan Regulation and Its Effects on Adhesion of Vascular Smooth Muscle Cells." Diss., Georgia Institute of Technology, 2005. http://hdl.handle.net/1853/7007.
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An injured vascular system has a substantial impact on an individuals overall health, and an understanding of the mechanisms that underlie blood vessel pathophysiology is required for the development of rational and effective treatment strategies. The phenotypic modulation of smooth muscle cells (SMC) during vascular injury, characterized by altered adhesion, migration and synthetic behavior, plays an important role in the eventual outcome. Specifically, the ability of SMCs to adhere to and remodel their extracellular environment via regulation of the syndecan class of cell adhesion molecules dictates the response of the vascular wall to local injury. The effect of in vitro syndecan-4 regulation on SMC adhesion was investigated through the use of a glass microsphere centrifugation assay, and an antisense-mediated reduction in gene expression was found to correlate with decreased adhesive strength. Regulation of syndecan-1, syndecan-2, and syndecan-4 gene expression was observed experimentally by mechanical strain of SMCs. Using real-time polymerase chain reaction (PCR), the kinetics of both static and cyclic mechanical strain were found to modify the gene expression in a time and strain magnitude-dependent manner unique to each syndecan. In particular, the responses of syndecan-4 were acute, but transient, while the evolution of syndecan-1 and syndecan-2 regulation was delayed by comparison. Mechanical strain also modulated syndecan-4 protein expression and ectodomain shedding, as measured by Western immunoblotting, and this effect was found, through selective inhibition, to be at least in part dependent on mitogen-activated protein (MAP) kinase signaling. In particular, intact extracellular signal-regulated MAP kinase (ERK) 1/2 and c-Jun NH2-terminal kinase / stress-activated protein kinase (JNK/SAPK) signaling pathways were found to be required for the observed strain-induced shedding. These findings offer a better understanding of syndecan function in response to mechanical strain and suggest potential new mechanisms by which physical forces may modulate vascular SMC behavior and regulation during normal physiology, pathologic conditions, and engineered arterial substitute development.
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Maitra, Sushmit. "The AU-rich element mRNA decay-promoting activity of BRF1 is regulated by mitogen-activated protein kinase activated protein kinase 2." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2008r/maitra.pdf.
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Penfold, Lucy. "Investigating the roles of AMP-activated protein kinase and calcium/calmodulin-dependent protein kinase kinase β in prostate cancer." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/54390.
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Prostate cancer cells are characterised by rapid growth, proliferation and migration, which requires rewiring of cellular metabolism including increased lipid and protein synthesis. AMP-activated protein kinase (AMPK) is a conserved master regulator of energy homeostasis and acts to downregulate anabolism and cell growth, whilst upregulating catabolism to maintain cellular ATP levels. Whether these actions inhibit or aid cancer progression is controversial. Intriguingly, an upstream activating kinase of AMPK, calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) has recently been implicated in prostate cancer progression. A small-molecule direct activator of AMPK, 991, was used to test the effects of AMPK activation in a panel of prostate cancer cell lines. AMPK activation led to downregulation of cellular proliferation, 2D migration, invasion and lipogenesis, and upregulation of adhesion in all cell lines. However, in PC3 and 22RV1 cells AMPK activation led to an increase in migration down a chemoattractant gradient. This increase in migration was dependent on CaMKKβ and PAK1 activity. To investigate the role of AMPK and CaMKKβ in vivo the PTEN prostate cancer mouse model was used. AMPKβ1 and CaMKKβ were deleted in this model creating two novel mouse lines. Upon β1-deletion prostate cancer development was increased based on the timing of a switch in protein expression characterised in the PTEN-/- prostate upon disease progression and pathological analysis of tissue sections. In contrast, disease progression was significantly reduced upon CaMKKβ-deletion in the PTEN-/- prostate based on prostate weight, Ki-67 staining and pathological analysis. Disease progression was also inhibited in the PTEN mouse model upon treatment with a pharmacological inhibitor of CaMKKβ, STO609. These data suggest that AMPK and CaMKKβ have different roles in prostate cancer development and progression likely lie on separate pathways in this disease.
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Nadal, Ribelles Mariona 1984. "Control of transcription by the stress activated Hog1 kinase." Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/293257.
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A fundamental property of living cells is the ability to sense and robustly respond to fluctuations in their environment. In budding yeast (Saccharomyces cerevisiae) changes in extracellular osmolarity are sensed by the HOG pathway, which evokes the program for cell adaptation required for cell survival.
The aim of this thesis was to further characterize the molecular mechanisms by which Hog1 regulates gene expression upon osmostress. A genome-wide genetic screen lead to the identification of several activities required for regulation of gene expression. Here we describe the characterization of a novel substrate for the SAPK whose activity is required for proper transcription initiation and elongation in response to stress.
This thesis also aimed to globally characterize the role of Hog1 in reprogramming the transcriptome of S. cerevisiae under osmostress conditions. By the combination of molecular approaches coupled to genome-wide techniques (ChIP-seq, MNase-seq and Tiling arrays) we have been able to fully characterize the localization of the key components that drive osmoresponsive transcription, providing for the first time a complete picture of the transcription process. The high resolution of the genome-wide approaches, has allowed us to identify new transcriptional roles for the SAPK such as the targeting of RNA Pol III machinery, and the regulation of a novel class of functional long noncoding RNAs (lncRNA). In summary, results presented in this thesis provide novel insights into the mechanisms by which the Hog1 SAPK modulates gene expression.Una propietat cel·lular fonamental és l’habilitat de detectar i respondre de forma robusta a les fluctuacions en el seu entorn. En cèl·lules de llevat (Saccharomyces cerevisiae), els canvis en l’ osmolaritat extracel·lular són detectats per la via de senyalització de HOG, que coordina el procés d’adaptació cel·lular imprescindible per sobreviure a un estrès osmòtic. L’objectiu d’aquest estudi és identificar i caracteritzar els mecanismes moleculars utilitzats per Hog1 per regular l’expressió gènica en resposta a estrès osmòtic. Fent servir un crivatge genètic a gran escala dissenyat per identificar activitats necessàries per la regulació de l’expressió gènica en resposta a estrès osmòtic, hem identificat un nou substrat de Hog1, l’activitat del qual és requereix tan per la iniciació com l’ elongació de la transcripció. En aquest treball també ens hem centrat en caracteritzar el paper global de Hog1 en la reorganització del transcriptoma de S. cerevisae en condicions d’ estrès osmòtic. Mitjançant la combinació de tècniques moleculars amb tècniques de seqüenciació (ChIP-seq, MNase-seq, Tiling arrays) hem definit el posicionament en el genoma dels components claus que regulen la transcripció, oferint per primera vegada una visió general del procés de transcripció en resposta a estrès osmòtic L’alta resolució d’aquestes tècniques ens ha permès identificar noves dianes transcripcionals de Hog1, com és la regulació d’una altra maquinària transcripcional (RNA Pol III) i la regulació de la transcripció de una nova classe de RNAs no codificants (lncRNAs). En conjunt, els resultats presentats en aquesta tesi proporcionen una nova visió dels mecanismes per els quals Hog1 modula l’expressió gènica
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Türk, Roland Daniel. "Expanding the signaling network around AMP-activated protein kinase /." Zürich : ETH, 2007. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17222.
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Zeng, Qingning. "Dissecting mitogen-activated protein kinase cascades involving arabidopsis MKK6." Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/31941.
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Mitogen-activated protein (MAP) kinase cascades act as critical components in the signalling pathways of all eukaryotic cells. They play a pivotal role in the transduction of extra- and intra-cellular stimuli and regulate cell growth, proliferation, differentiation and cell death, through sequential activation of MAP kinase kinase kinases (MAPKKKs), MAP kinase kinases (MKKs), and MAP kinases (MPKs). These three components form modules that control the phosphorylation of various substrates including transcription factors, enzymes, and cytoskeleton-associated proteins. In the Arabidopsis genome, over 60 MAPKKKs (AtMKKK), 10 MAPKKs (AtMKK), and 20 MAPKs (AtMPK) have been identified. The smaller number of AtMKKs suggests that diverse signals may converge and be integrated at the level of AtMKK. Among the ten AtMKKs, MKK6 has been proposed to play a role in regulating cytokinesis. However, little is known about the hierarchal phosphorylation system containing MKK6.
In this Ph.D. project, I aimed to dissect the MAP kinase cascades involving MKK6 in Arabidopsis. I investigated potential targets of MKK6. Four MAP kinases were identified to interact with, and be phosphorylated by, MKK6, namely, MPK4, MPK6, MPK11, and MPK13. Among them, MPK13 is developmentally co-expressed with MKK6, and both MPK13 and MKK6 display high Promoter::GUS activity at the primary root tips and at the lateral root primordia. Partial suppression of either MKK6 or MPK13 expression significantly reduces the number of lateral roots in the transgenic lines, suggesting that the MKK6-MPK13 module positively regulates lateral root formation.
Loss-of-function mutants of another potential target of MKK6, MPK4, are severely affected. In mpk4 mutant plants, anthers can develop normal microspore mother cells (MMCs) and peripheral supporting tissues, but the MMCs fail to complete meiotic cytokinesis, a phenotype reminiscent of those observed in both atnack2/tes/stud and anq1/mkk6 mutants. Biochemical analysis defines a putative signalling module linking AtNACK2/TES/STUD with ANP3, MKK6 and MPK4, suggesting a model in which the AtNACK2-ANP3-MKK6-MPK4 cascade specifically facilitates male meiotic cytokinesis in Arabidopsis.
In summary, my PhD work has expanded our understanding of the diversity of MAP kinase cascades. Different MPKs can act as downstream targets of the same MKK with tight spatial and temporal regulation during plant development.