National Repository of Grey Literature 12 records found  1 - 10next  jump to record: Search took 0.00 seconds. 
Structural characterization of human protein kinase CaMKK2 and its interactions with binding partners
Koupilová, Nicola ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
5 Abstract Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) belongs to the serine/ threonine protein kinase family, which is involved in the calcium signaling pathway. The increase of intracellular calcium concentration induces the activation of calmodulin (CaM), which then activates its binding partners including CaMKII, CaMKIII, CaMKK1 and CaMKK2. CaMKK2 activates CaMKI, CaMKIV and AMP-dependent kinase, AMPK, by phosphorylation. CaMKK2 is naturally present in cells in an autoinhibited state, which is caused by the steric hindrance of the active site by the autoinhibitory domain. When calmodulin binds to the calmodulin-binding domain, the autoinhibitory domain is removed and the active site becomes accessible. Upon activation, CaMKK2 undergoes autophosphorylation, which increases its enzyme activity. Negative regulation of CaMKK2 is mediated by cAMP-dependent protein kinase A (PKA)- and GSK3-dependent phosphorylation. Sites phosphorylated by PKA have been identified for both CaMKK1 and CaMKK2. Two of them are also motifs recognized by scaffolding 14-3-3 proteins. Previous studies have shown that the 14-3-3 protein binding maintains phosphorylated CaMKK2 in an inhibited state by blocking the dephosphorylation of S495, which prevents the binding to calmodulin. However, it is unclear if it is the...
The role of ERK1 and ERK2 protein kinases in the MAPK/ERK signaling
Galvánková, Kristína ; Vomastek, Tomáš (advisor) ; Dráber, Peter (referee)
The MAPK/ERK cascade is highly conserved signalling pathway regulating cellular processes which are necessary for cell life, such as proliferation, differentiation, apoptosis or cell migration. All these cellular responses are the result of the processing of extracellular signals through three-tier ERK cascade consisting of protein kinases Raf, MEK and ERK. The signal is transmitted by sequential phosphorylation where RAF phosphorylates MEK and MEK phosphorylates and activates ERK. Protein kinase ERK then phosphorylates and regulates a wide range of substrates at different locations in the cell. This affects the cellular response to the extracellular signal. Regulation of this pathway on every level is very important and is modulated by interaction partners and adaptor proteins. Deregulation of the pathway as well as mutations of individual protein kinases can lead to severe pathological consequences. At the level of ERK, there are two isoforms, ERK1 and ERK2, which are more than 80 % identical at the amino acid level. Their high sequence similarity has triggered the interest of many authors for more detailed examination of both isoforms in respect of their evolutionary conservation and whether they are functionally redundant or whether they have specific functions. The aim of this work is to...
Study of regulatory mechanisms of selected protein kinases
Petrvalská, Olívia
Through binding interactions with more than 300 binding partners, 14-3-3 proteins regulate large amount of biologically relevant processes, such as apoptosis, cell cycle progression, signal transduction or metabolic pathways. The research discussed in this dissertation thesis was focussed on investigating the role of 14-3-3 proteins in the regulation of two selected protein kinases ASK1 and CaMKK2. The main goal was to elucidate the mechanisms by which phosphorylation and 14-3-3 binding regulate functions of these protein kinases using various biochemical and biophysical methods, such as site-directed mutagenesis, enzyme activity measurements, analytical ultracentrifugation, small-angle X-ray scattering, chemical crosslinking, nuclear magnetic resonance and fluorescence spectroscopy. A structural model of the complex between the catalytic domain of protein kinase ASK1 with 14-3-3ζ, which was calculated using the small-angle X-ray scattering and chemical crosslinking data, suggested that this complex is conformationally heterogeneous in solution. This structural model together with data from time-resolved fluorescence and nuclear magnetic resonance suggested that the 14-3-3ζ protein interacts with the catalytic domain of ASK1 in the close vicinity of its active site, thus indicating that the complex...
Preparation of human Ca2+/calmodulin-dependent protein kinase kinase 2 phosphorylated at Ser100 and Ser511
Koupilová, Nicola ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
5 Abstract Ca2+ /calmodulin-dependent protein kinase kinases (CaMKK) are serine/threonine kinases involved in the calcium signaling pathway. Two CaMKK isoforms were described in mammals: CaMKK1 and CaMKK2. The increase in calcium concentrations induces Ca2+ /CaM binding to the C-terminal segment of CaMKK, thus relieving autoinhibition by disrupting the interaction between the autoinhibitory segment and the kinase domain. Active CaMKK then phosphorylate and activate their downstream kinases CaMK1 and CaMK4, and in the case of CaMKK2 also AMPK. The activity of CaMKK is also regulated by phosphorylation mediated by cAMP-dependent protein kinase A (PKA). This phosphorylation creates two binding motifs recognized by the regulatory 14-3-3 proteins. Previous studies have suggested that the 14-3-3 protein keeps phos- phorylated CaMKK1 in the inhibited state by blocking the dephosphorylation of the inhibitory phosphorylation site and it has been speculated that CaMKK2 is regulated in a similar manner. However, the role of 14-3-3 protein in the regulation of CaMKK2 is unclear. In order to study this protein complex, it is necessary to prepare recombinant CaMKK2 fully phosphorylated at both 14-3-3 binding motifs. The main aim of this bachelor thesis was to optimize the protocol for the phosphorylation of human CaMKK2...
Study of regulatory mechanisms of selected protein kinases
Petrvalská, Olívia
Through binding interactions with more than 300 binding partners, 14-3-3 proteins regulate large amount of biologically relevant processes, such as apoptosis, cell cycle progression, signal transduction or metabolic pathways. The research discussed in this dissertation thesis was focussed on investigating the role of 14-3-3 proteins in the regulation of two selected protein kinases ASK1 and CaMKK2. The main goal was to elucidate the mechanisms by which phosphorylation and 14-3-3 binding regulate functions of these protein kinases using various biochemical and biophysical methods, such as site-directed mutagenesis, enzyme activity measurements, analytical ultracentrifugation, small-angle X-ray scattering, chemical crosslinking, nuclear magnetic resonance and fluorescence spectroscopy. A structural model of the complex between the catalytic domain of protein kinase ASK1 with 14-3-3ζ, which was calculated using the small-angle X-ray scattering and chemical crosslinking data, suggested that this complex is conformationally heterogeneous in solution. This structural model together with data from time-resolved fluorescence and nuclear magnetic resonance suggested that the 14-3-3ζ protein interacts with the catalytic domain of ASK1 in the close vicinity of its active site, thus indicating that the complex...
Biophysical characterization of the N-terminal part of protein kinase ASK1.
Honzejková, Karolína ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
Apoptosis signal-regulating kinase 1 (ASK1) is an apical kinase of the mitogen-activated protein kinase cascade. Its activity is triggered by various stress stimuli such as reactive oxygen species (ROS), cytokines, endoplasmic reticulum (ER) stress or osmotic stress resulting in the activation of p38 and c-Jun N-terminal kinase metabolic pathways and leading to inflammation or cell death. Dysregulation of ASK1 is linked to several pathologies such as neurodegenerative and cardiovascular diseases and cancer, which makes this protein a potential target of therapeutic intervention. The activity of ASK1 is regulated through protein-protein interactions with 14-3-3 proteins and thioredoxin1 being among the most important negative regulators and tumour necrosis factor receptor-associated factors being an example of positive regulators. Apart from that, ASK1 is also tightly regulated via oligomerization. Despite continual progress being made, the precise molecular mechanism of ASK1 regulation and the role of ASK1 oligomerization in this process still remains unclear to this day owing to the lack of structural data. Interaction of the N-terminal parts of two protomers of ASK1 dimer is one of the key steps in ASK1 activation. It was shown, that the isolated ASK1 catalytic domain (ASK1-CD) forms stable...
Structural studies of selected signaling protein complexes.
Pšenáková, Katarína ; Obšil, Tomáš (advisor) ; Hrabal, Richard (referee) ; Maloy Řezáčová, Pavlína (referee)
The ability of proteins to bind other molecules in response to various stimuli in their microenvironment serves as a platform for extensive regulatory networks coordinating downstream cell actions. The correct function of these signaling pathways depends mostly on noncovalent interactions often affecting the structure of proteins and protein complexes. Understanding the molecular mechanism of a protein function in cell signaling therefore often depends on our knowledge of a three-dimensional structure. In this doctoral thesis, I present the work that led to the understanding of several protein-protein and protein-ligand interactions implicated in cell signaling at the molecular level. I applied nuclear magnetic resonance spectroscopy, small angle X-ray scattering and other biophysical methods to determine the molecular basis of inhibition of four signaling proteins: Calcium/Calmodulin (Ca2+ /CaM)-dependent protein kinase kinase 2 (CaMKK2); protease Caspase-2; Forkhead transcription factor FOXO3, and Apoptosis signal-regulating protein kinase 1 (ASK1). In particular, I investigated the distinct roles of 14-3-3 and Ca2+ /CaM in the regulation of CaMKK2 activity. I also studied in detail the mechanism how 14-3-3 interferes with the caspase-2 oligomerization and its nuclear localization as well as...
Study of regulatory mechanisms of selected protein kinases
Petrvalská, Olívia ; Obšil, Tomáš (advisor) ; Jiráček, Jiří (referee) ; Schneider, Bohdan (referee)
Through binding interactions with more than 300 binding partners, 14-3-3 proteins regulate large amount of biologically relevant processes, such as apoptosis, cell cycle progression, signal transduction or metabolic pathways. The research discussed in this dissertation thesis was focussed on investigating the role of 14-3-3 proteins in the regulation of two selected protein kinases ASK1 and CaMKK2. The main goal was to elucidate the mechanisms by which phosphorylation and 14-3-3 binding regulate functions of these protein kinases using various biochemical and biophysical methods, such as site-directed mutagenesis, enzyme activity measurements, analytical ultracentrifugation, small-angle X-ray scattering, chemical crosslinking, nuclear magnetic resonance and fluorescence spectroscopy. A structural model of the complex between the catalytic domain of protein kinase ASK1 with 14-3-3ζ, which was calculated using the small-angle X-ray scattering and chemical crosslinking data, suggested that this complex is conformationally heterogeneous in solution. This structural model together with data from time-resolved fluorescence and nuclear magnetic resonance suggested that the 14-3-3ζ protein interacts with the catalytic domain of ASK1 in the close vicinity of its active site, thus indicating that the complex...
Study of interactions between protein kinase CaMKK2 and calmodulin using fluorescence spectroscopy.
Mikulů, Martina ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
Ca2+ /calmodulin-dependent kinases are members of CaMK family, which is involved in CaMK cascade. One of CaMK family members is Ca2+ /calmodulin-dependent kinase kinase 2 (CaMKK2), which is activated by Ca2+ /CaM-binding. There are some structural differences between CaMKK2 and other protein kinases, one of them is a structure near αE-helix and autoinhibitory domain. Due to the overlap of autoinhibitory domain and Ca2+ /CaM-binding domain it can be supposed that Ca2+ /CaM-binding induces structural changes near autoinhibitory do- main and thus can affect the accessibility of this region. CaMKK2 W445F mutant, which contains only one tryptophane residue Trp374 close to the αE-helix, was expressed and purified. Structural changes in this region were monitored using tryptophan fluorescence intensity quenching experiments, which can provide information about the accessibility of region surrounding tryptophan residue. The fluorescence of Trp374 was quenched using acrylamide. Comparison of fluorescence quenching experiments performed in the presence and absence of calmodulin suggests that the complex formation induces structural change in the region surrounding Trp374 . 1
Role of Polo-like kinases in the cell cycle and DNA damage response
Kudláčková, Radmila ; Macůrek, Libor (advisor) ; Šolc, Petr (referee)
Within the process of cell division, genetic material must be equally distributed between the two daughter cells. In the next phase, the missing portion of the genome must be synthesized. The entire cycle is regulated by cyclin-dependent kinases (Cdks) in cooperation with cyclins. If the DNA is damaged during the cell cycle, signaling pathways of checkpoints supress cycle progression and enforce the cell cycle arrest until the damage is repaired. Malfunction of the checkpoints results in tumorigenesis. Polo-like kinases (Plks) are, much like Cdks, important regulators of the cell cycle. Plks play significant role mainly in the mitosis and also in a response to the DNA damage. This thesis is focused on human homologues, nevertheless conservation of homologues among organisms is considerable, thus presented findings are of general relevance. Human cells express five proteins from the family of Polo-like kinases, from which Plk1 corresponds to Polo-like kinases of lower eukaryotes. Knowledge on the remaining four kinases is still on the rise.

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