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In search of DUSP specificity
Sladeček, Stanislava ; Novotný, Marian (advisor) ; Martínková, Natália (referee)
Dual specificity phosphatases (DUSP) are enzymes that dephosphorylate both phosphoserine/threonine and phosphotyrosine residues on one substrate. Most of them specifically dephosphorylate family mitogen-activated protein kinases (MAPK). Number of DUSPs increases with complexity of organisms and in human genome there are 25 DUSPs described. Some DUSPs can dephosphorylate only one protein while other interact with wider spectrum of substrates. Except for substrate specificity DUSPs differ in expression, subcellular localization etc. Although first DUSPs were described about 20 years ago, a clear factor responsible for their substrate specificity is not known. This works uses in silico methods to discover and describe similarities and differences between DUSPs which may be important in determining DUSP specificity. Key words: phosphatase, kinase, DUSP, MAPK, substrate specificity, conservation of residues, phylogenetic tree, in silico methods
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The role of protein phosphorylation during progamic phase of tobacco male gametophyte development
Fíla, Jan
v angličtině (English abstract) Tobacco male gametophyte has a strongly dehydrated cytoplasm and represents a metabolically inactive stage. Upon cytoplasm rehydration, pollen grain becomes metabolically active and after the activation is finished, the pollen tube growth through a selected pollen aperture starts. The rehydration together with metabolic activation are accompanied by the regulation of translation and post-translational modifications (mainly phosphorylation) of the existing proteins. In this Ph.D. thesis, there were identified phosphopeptides from tobacco (Nicotiana tabacum) mature pollen, pollen activated in vitro 5 min and pollen activated in vitro 30 min. The total proteins from the above male gametophyte stages were extracted. The protein extract was trypsinized and the acquired peptide mixture was enriched by MOAC (metal oxide/hydroxide affinity chromatography) with titanium dioxide matrix. The enriched fraction was subjected to liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS). Totally, there were identified 471 phosphopeptides, carrying 432 exactly localized phosphorylation sites. The acquired peptide identifications were mapped to 301 phosphoproteins that were placed into 13 functional categories, dominant of which were transcription, protein synthesis,...
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Elucidation of ERK1 and ERK2 protein kinases effect on cap-independent translation initiation
Přibyl, Miroslav ; Vopálenský, Václav (advisor) ; Vomastek, Tomáš (referee)
Protein kinases ERK1 and ERK2 are one of the most studied proteins in cell signalling. Both proteins are involved in a plethora of processes, such as phosphorylation and activation of kinases as part of signalling pathways. Enzymes ERK1 and ERK2 are part of MAPK/ERK signalling cascade, connected to many cellular including cell proliferation, cell growth or differentiation. The MAPK/ERK signalling cascade is often activated in different types of tumors, making it a candidate for developing new chemical inhibitors. One of the important questions in fundamental research of ERK1 and ERK2 protein kinases is the search for difference between these proteins. Current knowledge points to redundancy of both proteins, howver several examples suggest otherwise. Recently, the work presented in Casanova et al. 2012 indirectly suggests divergent effect of ERK1 and ERK2 on cap-independent translation initiation. In the Laboratory of RNA biochemistry we focus on HCV IRES (Hepatitis C Virus Internal Ribosome Entry Site) dependent translation initiation. This diploma thesis lead to establish RNA interference method in our laboratory and to establish reporter system to study ERK1 and ERK2 effect on HCV IRES dependent translation initiation. Based on our data acquired during our research, we present in this work...
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The role of protein phosphorylation during progamic phase of tobacco male gametophyte development
Fíla, Jan ; Honys, David (advisor) ; Paleček, Jan (referee) ; Smýkal, Petr (referee)
v angličtině (English abstract) Tobacco male gametophyte has a strongly dehydrated cytoplasm and represents a metabolically inactive stage. Upon cytoplasm rehydration, pollen grain becomes metabolically active and after the activation is finished, the pollen tube growth through a selected pollen aperture starts. The rehydration together with metabolic activation are accompanied by the regulation of translation and post-translational modifications (mainly phosphorylation) of the existing proteins. In this Ph.D. thesis, there were identified phosphopeptides from tobacco (Nicotiana tabacum) mature pollen, pollen activated in vitro 5 min and pollen activated in vitro 30 min. The total proteins from the above male gametophyte stages were extracted. The protein extract was trypsinized and the acquired peptide mixture was enriched by MOAC (metal oxide/hydroxide affinity chromatography) with titanium dioxide matrix. The enriched fraction was subjected to liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS). Totally, there were identified 471 phosphopeptides, carrying 432 exactly localized phosphorylation sites. The acquired peptide identifications were mapped to 301 phosphoproteins that were placed into 13 functional categories, dominant of which were transcription, protein synthesis,...
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Study of interaction between ASK1 kinase and thioredoxin.
Koláčková, Kateřina ; Obšil, Tomáš (advisor) ; Vaněk, Ondřej (referee)
MAP kinase signaling cascade plays an important role in the cellular response to various stress stimuli from the external environment. This signaling cascade is divided into three levels: MAP kinase kinase kinases (MAP3K) phosphorylate and thus activate MAP kinase kinases (MAP2K) and those subsequently phosphorylate and thus activate MAP kinase (MAPK) pathway, which regulates many cellular functions such as apoptosis, cell differentiation and morphogenesis. One of the important MAP3K is protein kinase ASK1 (Apoptosis signal-regulating kinase 1), which is an important regulator of cellular immune and stress responses. Given that the increased activity of ASK1 is related to the development of serious diseases such as cancer, cardiovascular and neurodegenerative diseases, ASK1 is an interesting target in the pharmacy in the development of new drugs. Human ASK1 consists of 1374 amino acids and is divided into three domains: a central Ser/Thr catalytic domain and two coiled-coil domains, of which the first is located at the N- and the second at the C-terminus of the molecule of this protein kinase. ASK1 is regulated by its binding partners, which include a small cellular redox protein thioredoxin (Trx-1), which binds to the N-terminal part of ASK1. Trx-1 is a potent antioxidant and so it protects cells...
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Preparation and characterization of the catalytic domain of human protein kinase ASK1.
Petrvalská, Olívia ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
Protein kinase ASK1 (apoptosis signal-regulating kinase 1) is a member of the mitogen- activated protein kinase kinase kinase (MAP3K) family and plays a crucial role in immune and stress responses. Since the increased activity of ASK1 has been linked to the development of several diseases including cancer, cardiovascular and neurodegenerative diseases, this enzyme is a promising target for therapeutical intervention in these pathologies. The molecule of ASK1 consists of 1374 amino acid residues, but catalytic activity possesses only a kinase domain located approximately in the middle of the molecule. The activity of ASK1 is regulated by interactions with various proteins including the 14-3-3 protein. This protein recognizes a phosphorylated motif around Ser966 at the C-terminus of the catalytic domain of ASK1. This binding interaction inhibits ASK1 through unknown mechanism. ASK1 under stress conditions, such as oxidative stress, is dephosphorylated at Ser966 and the 14-3-3 protein dissociates. This dissociation is then one of the factors that lead to the activation of ASK1. The aim of this diploma thesis was to prepare a complex of the catalytic domain of ASK1 with the 14-3-3 protein for subsequent structural studies. Both proteins were expressed in E. coli cells and successfully purified. In...
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ERK1/2 MAP kinase - Structure and Interaction Partners
Přibyl, Miroslav ; Vopálenský, Václav (advisor) ; Žíla, Vojtěch (referee)
Extracellular signal molecules are recognized by membrane receptors on the surface of eukaryotic cells. Receptors transmit the signal into the intracellular space where activation of the concrete enzymes occurs. Activated enzymes may be protein kinases that phosphorylate the substrate proteins corresponding to the requirements for specific recognition by a protein kinase. Substrate proteins may be structural proteins and enzymes, which in turn transmit the signal or directly affect the physiological processes of the cell. The protein kinase family accounts for ERK1 and ERK2 (Extracellular signal-regulated kinase 1 and 2), enzymes which affect cell growth, cell cycle and numerous other physiological processes of the cell. Protein kinases are dynamic molecules, which undergo a series of conformational changes during their catalytic cycle and whose stability and function are affected by conformational changes. Conserved amino acid residues carry out the function of protein kinases. These factors are also involved in interactions with protein substrates and regulatory proteins, and are responsible for specific function of protein kinase.
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