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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...
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Structural and regulatory aspects of Src kinase activation
Koudelková, Lenka
Src kinase plays a crucial role in a multitude of fundamental cellular processes. Src is an essential component of signalling pathways controlling cellular proliferation, motility or differentiation, and is often found deregulated in tumours. Src activity is therefore maintained under stringent and complex regulation mediated by SH3 and SH2 domains and the phosphorylation state of tyrosines 416 and 527. Active Src adopts an open conformation whereas inactive state of the kinase is characterised by a compact structure stabilised by inhibitory intramolecular interactions. We identified phosphorylation of tyrosine 90 within binding surface of SH3 domain as a new regulatory switch controlling Src kinase activation. Using substitutions mimicking phosphorylation state of the residue we demonstrated that tyrosine 90 phosphorylation controls Src catalytic activity, conformation and interactions mediated by the SH3 domain, representing a positive regulatory mechanism leading to elevated activation of mitogenic pathways and increased invasive potential of cells. Based on correlation between compactness of Src structure and its catalytic activity, we constructed a FRET-based sensor of Src conformation enabling to measure the dynamics of Src activation in cells with spatio-temporal resolution. We found that...
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Structural and regulatory aspects of Src kinase activation
Koudelková, Lenka
Src kinase plays a crucial role in a multitude of fundamental cellular processes. Src is an essential component of signalling pathways controlling cellular proliferation, motility or differentiation, and is often found deregulated in tumours. Src activity is therefore maintained under stringent and complex regulation mediated by SH3 and SH2 domains and the phosphorylation state of tyrosines 416 and 527. Active Src adopts an open conformation whereas inactive state of the kinase is characterised by a compact structure stabilised by inhibitory intramolecular interactions. We identified phosphorylation of tyrosine 90 within binding surface of SH3 domain as a new regulatory switch controlling Src kinase activation. Using substitutions mimicking phosphorylation state of the residue we demonstrated that tyrosine 90 phosphorylation controls Src catalytic activity, conformation and interactions mediated by the SH3 domain, representing a positive regulatory mechanism leading to elevated activation of mitogenic pathways and increased invasive potential of cells. Based on correlation between compactness of Src structure and its catalytic activity, we constructed a FRET-based sensor of Src conformation enabling to measure the dynamics of Src activation in cells with spatio-temporal resolution. We found that...
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The role of inflammatory signaling in cancer cell invasiveness
Šůchová, Anna-Marie ; Brábek, Jan (advisor) ; Brdička, Tomáš (referee)
Metastasizing is responsible for 90% of death in cancer patients. Metastatic tumour cells have several strategies that they use to invade surrounding tissues - they can migrate together or individually. When individual cells migrate, tumour cells adopt two different morphologies. They are either elongated and migrate using the proteolytically active mesenchymal mode, or they are rounded and migrate in the amoeboid mode. Metastatic tumour cells can switch between these modes, which complicates the development of effective migrastatics. In this work, we focused on the effect of inflammatory signalling on metastatic cell migration. We worked with cell lines of malignant human melanoma, which adopt a mixed morphology and show both amoeboid and mesenchymal phenotype during migration. Upon stimulation of melanoma human cells with interferon beta, a mesenchymal to amoeboid transition occurs. Interferon beta appears to induce amoeboid morphology by maintaining high levels of the ISGF3 complex, which is composed of the heterodimer of STAT 1 and STAT 2 proteins and the IRF9 protein. Upon blocking of Jak / Stat signalling pathway by negative regulators, human melanoma cells return to mesenchymal morphology. Key words - invasiveness, mesenchymal-ameboid transition, interferons, inflammation, migration, metastases
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Structural and regulatory aspects of Src kinase activation
Koudelková, Lenka ; Brábek, Jan (advisor) ; Brdička, Tomáš (referee) ; Hejnar, Jiří (referee)
Src kinase plays a crucial role in a multitude of fundamental cellular processes. Src is an essential component of signalling pathways controlling cellular proliferation, motility or differentiation, and is often found deregulated in tumours. Src activity is therefore maintained under stringent and complex regulation mediated by SH3 and SH2 domains and the phosphorylation state of tyrosines 416 and 527. Active Src adopts an open conformation whereas inactive state of the kinase is characterised by a compact structure stabilised by inhibitory intramolecular interactions. We identified phosphorylation of tyrosine 90 within binding surface of SH3 domain as a new regulatory switch controlling Src kinase activation. Using substitutions mimicking phosphorylation state of the residue we demonstrated that tyrosine 90 phosphorylation controls Src catalytic activity, conformation and interactions mediated by the SH3 domain, representing a positive regulatory mechanism leading to elevated activation of mitogenic pathways and increased invasive potential of cells. Based on correlation between compactness of Src structure and its catalytic activity, we constructed a FRET-based sensor of Src conformation enabling to measure the dynamics of Src activation in cells with spatio-temporal resolution. We found that...
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The role of galectins in cancer cell invasiveness
Remišová, Michaela ; Brábek, Jan (advisor) ; Kovář, Marek (referee)
Galectins are family of β-galactosidase binding proteins that serve many functions in all kind of mammalian cells. In the past years galectins, namely galectin-1 and galectin-3, have been revealed to play a major role in various cancer processes including cancer cell invasiveness, a process indispensable for the formation of metastasis. Both extracellular and intracellular forms of galectins modify the process of invasiveness in various ways, through interacting with different components of the cell or of the cell signalling pathways. The aim of this bachelor's thesis is to summarize mechanisms by which galectins promote cancer cell invasiveness. Keywords: galectins, galectin-1, galectin-3, invasiveness, cancer, metastasis
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The Role of the Tumour Microenvironment on Melanoma Cell Invasiveness
Jobe, Njainday ; Rösel, Daniel (advisor) ; Kořínek, Vladimír (referee) ; Bušek, Petr (referee)
Cancer cell invasion and metastasis are hallmarks of cancer. It is becoming apparent that the interaction between cancer cells and the surrounding microenvironment are involved in their ability to invade and metastasise. In general, cancer cells can either migrate individually, in an amoeboid or mesenchymal manner, or collectively. The first aim of this thesis was to analyse the role of NG2 in amoeboid to mesenchymal transition (AMT) and Rho/ROCK signalling. We found that NG2 promotes an amoeboid morphology, and increased invasiveness, in a Rho-dependent manner. Secondly, we analysed the role of the major tumour microenvironment (TME) component, cancer-associated fibroblasts (CAFs), on melanoma cell invasiveness. We found the CAF interaction with melanoma cells leads to increased levels of interleukin-6 (IL-6) and IL-8, and this leads to increased invasiveness. Simultaneous blocking of IL-6 and IL-8, using neutralising antibodies, inhibits CAF-dependent invasion. Further analysis of another major component in the melanoma TME, keratinocytes, has highlighted the importance of the tumour cell niche in invasion. Our results indicate that cancer cells have the ability to change morphology, and that the TME plays an important role in melanoma cell invasiveness. Metastatic melanoma treatment has proven...
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The plasticity of melanoma cell invasiveness
Gandalovičová, Aneta ; Brábek, Jan (advisor) ; Truksa, Jaroslav (referee)
and keywords: During metastasis, cancer cells can invade the extracellular matrix using various strategies. When invading individually, they employ either the amoeboid invasion mode, during which the cell body dynamically deforms by enhanced contractility to squeeze through pores within the matrix, or protease dependent mesenchymal migration that takes advantage of the possibility to digest the surrounding matrix. Cells migrating in one mode can actively switch to the other by mesenchymal-amoeboid (MAT) or amoeboid-mesenchymal transitions (AMT). This enables escape mechanisms and considerably complicates anti-metastatic treatment. It is well known that Rho GTPases are master regulators of cytoskeleton re-arrangements and thus, unsurprisingly, play a major role in both invasion modes and can directly drive the transitions. However, upstream activation of these pathways is still largely unclear. This thesis aimed to optimize 3D conditions suitable for studying plasticity of cell invasion in vitro, establish AMT and MAT in melanoma cells based on manipulation of Rho GTPases and verify novel candidates regulating cell invasion plasticity based on previous RNA sequencing of cells before and after MAT. Last, by synthesis of published data, results from sequencing and new findings presented in this...
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The role of cell polarity signaling in the plasticity of cancer cell invasiveness
Gandalovičová, Aneta ; Brábek, Jan (advisor) ; Cvrčková, Fatima (referee)
Throughout the last few years cancer research has focused on studying the origin of secondary tumors, i.e. metastases, which are a direct outcome of the ability of cancer cells to disseminate from the primary tumor and invade the adjacent tissue. Generally, cancer cells migrate by two distinct mechanisms- amoeboid or mesenchymal. Whereas the mesenchymal migration mode can be described as "path generating", the amoeboid mode resembles a "path finding" way of migration. Both types of invasion are regulated by divergent signaling pathways that are closely related to cell polarity and cytoskeleton reorganization. Responsible for cell polarization are not only the polarity complexes Par, Scribble and Crumbs, but also phosphoinositides and Rho GTPases Rac, Rho and Cdc42, which, additionally, regulate the dynamics of the cytoskeleton. By a mutual interplay they regulate cell motility. It cannot come as a surprise that their deregulation commonly results in tumorigenesis. A more thorough comprehension of the signaling pathways leading to cancer cell invasiveness is a necessary step towards understanding the complex problem of metastasis. Key words: invasiveness, amoeboid, mesenchymal, cell polarity, motility, Rho GTPases, polarity complexes
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The role of proto-oncogene crk in invasiveness
Tomášová, Lea ; Rösel, Daniel (advisor) ; Ševčík, Jan (referee)
Proto-oncogene Crk was identified as an oncogenic product of an avian retrovirus in 1988. It is an adaptor protein containing SH2 and SH3 binding domains. Thanks to these domains Crk facilitates protein-protein interactions and therefore plays a crucial role in signal transduction. Crk forms signal complexes with several proteins and hence impacts many cellular processes, among them cell migration, tumorigenezis and invasion of the surrounding tissues. The increased invasiveness allows the tumour cells to detach from the primary tumour and form metastasis which is very problematic feature of cancer. Overexpression of Crk was observed in several tumour tissues, it correlates with an aggressive and metastatic phenotype of the tumours. The subject of this thesis is to describe the mechanisms of how Crk can regulate cellular motility and invasiveness.
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