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Functional analysis of the internally disordered structure of the substrate domain in p130Cas protein biology
Hejnarová, Marie ; Rösel, Daniel (advisor) ; Gemperle, Jakub (referee)
The p130Cas protein is an important mechanosensor in cell adhesive structures such as focal adhesions and podosomes. There, the protein is subjected to mechanical tension that underlie its participation in integrin signaling. At the level of p130Cas, the mechanical force is transduced into a chemical signal. Although p130Cas does not exhibit enzymatic activity, its high binding potential turns it into an important signaling junction that ensures signal distribution to different cellular pathways. As a result, p130Cas has a profound effect on fun- damental cellular processes such as cell proliferation, differentiation and motility. In addition to its irreplaceable role in embryonic development, its involvement in the origin of patholo- gies has also been reported. As a major substrate for Src kinase, p130Cas can participate in signaling leading to tumor transformation and further malignant development. Its upregulated expression is ofen observed in aggressive types of metastasizing tumors, such as breast, prostate, and melanoma cancer. Therefore, in recent years, the possible use of the p130Cas protein as a potential target for migrastatics under development has been discussed. Within this thesis, we focus on the functional analysis of the p130Cas substrate domain. This domain is respon- sible for...
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Characterization of perinuclear actin fibers and their role in cell migration
Hlaváčková, Tereza ; Vomastek, Tomáš (advisor) ; Binarová, Pavla (referee)
Cell migration is crucial for such physiological and pathological processes as wound healing, emryonal development, immune response, and methastasizing of the cancer cells. It is tightly coupled with cell polarization, nuclear traslocation, and turnover of actin cytoskeleton. Substantial, but so far poorely explored, part of actin cytoskeleton is perinuclear actin cap - dome-like structure above the nucleus costructed from perinuclear actin fibers. At the apical side of the nucleus perinuclear actin fibers are associated with LINC complex through nesprin proteins; at the edges of the cell they are anchored to focal adhesions. In the literature there were assumptions that this type of actin fibers can generate traction forces for nuclear reorientation during cell migration. The aim of this thesis is to elucidate the mechanism involved in the attachment of perinuclear actin to the LINC complex and the nucleus, thereby regulating the formation of the perinuclear actin cap. In addition, we aimed to establish a semi- automatic tool for perinuclear actin fibers quantification. Rat2 fibroblasts were used as the model cell line because they contain well-developed perinuclear actin cap. We focused on the inactivation of LINC complex components, namely Giant nesprin proteins (nesprin 1 and nesprin 2) and...
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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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Construction and evaluation of a novel protein mechanosensor
Kolomazníková, Veronika ; Rösel, Daniel (advisor) ; Novotný, Ivan (referee)
The protein p130Cas (human ortholog BCAR1) is a major substrate for phosphorylation by the Src family kinase and plays a central role in oncogenic transformation. Increased level of BCAR1 correlates with primary tumour growth and cancer progression. Localized to focal adhesion, p130Cas serves as a mechanosensor and mediates key interactions with the extracellular environment. The structure of p130Cas is crucial for its function, mainly the anchoring domains SH3 and CCH, together with the substrate domain which is extended when under tension. This Master's thesis presents a newly developer FRET mechanosensor based on the structure of p130Cas. The sensor utilizes the anchoring domains of p130Cas for proper localization to focal adhesions, where it can detect tension in living cells. Key words: p130CAS, FRET, focal adhesions, mechanosensing
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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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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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Construction and evaluation of a novel protein mechanosensor
Kolomazníková, Veronika ; Rösel, Daniel (advisor) ; Novotný, Ivan (referee)
The protein p130Cas (human ortholog BCAR1) is a major substrate for phosphorylation by the Src family kinase and plays a central role in oncogenic transformation. Increased level of BCAR1 correlates with primary tumour growth and cancer progression. Localized to focal adhesion, p130Cas serves as a mechanosensor and mediates key interactions with the extracellular environment. The structure of p130Cas is crucial for its function, mainly the anchoring domains SH3 and CCH, together with the substrate domain which is extended when under tension. This Master's thesis presents a newly developer FRET mechanosensor based on the structure of p130Cas. The sensor utilizes the anchoring domains of p130Cas for proper localization to focal adhesions, where it can detect tension in living cells. Key words: p130CAS, FRET, focal adhesions, mechanosensing
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The role of p130CAS in integrin signaling
Janoštiak, Radoslav ; Brábek, Jan (advisor) ; Brdička, Tomáš (referee) ; Kořínek, Vladimír (referee)
Focal adhesions are important subcellular structures that are composed of many signaling and scaffolding proteins. They serve not only for anchoring the cell to the substratum but they are also important signaling centers that regulate various cellular behavior such as migration, invasiveness, proliferation and survival. Focal adhesion signaling needs to be strictly regulated because alteration in activity or expression of many focal adhesion proteins leads to tumorogenesis and metastasis formation. One of the most important scaffolding protein associated with focal adhesion is p130Cas. The importance of p130Cas in regulation of cell migration and invasiveness has been well established. P130Cas also plays important role in regulation of cell survival and proliferation. Moreover, high protein levels of human ortholog of p130Cas - BCAR1, has been linked to more aggressive breast tumors and poor prognosis. During my doctoral studies, I focused on the role of p130Cas in integrin signaling. At the beginning we characterized the role of tyrosine 12 phosphorylation within its SH3 domain. We confirmed that this phosphorylation is increased in Src527F transformed mouse embryonic fibroblasts compared to non-transformed counterparts and also in some human cancer cell lines. We showed that this phosphorylation...
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The regulation of the ERK signalling pathway by scaffold protein RACK1
Bráborec, Vojtěch ; Vomastek, Tomáš (advisor) ; Filipp, Dominik (referee)
The ERK signalling cascade comprised of protein kinases Raf, MEK and ERK is an evolutionarily conserved member of MAPK family that is activated in response to wide range of extracellular stimuli. The ERK pathway controls fundamental cellular functions including cell proliferation, differentiation, apoptosis or cell motility. To control such a diverse cellular responses by a single pathway cells have evolved regulatory mechanisms that channel the extracellular signals towards the specific biological response. Crucial to this control are non- enzymatic proteins termed scaffolds that associate with and enhance functional interaction of the components of MAPK pathways and can regulate amplitude, timing, specificity and location of signals. Scaffold protein RACK1 associates with several components of cell migration machinery including integrins, FAK, Src and the ERK pathway core protein kinases. RACK1 regulates distinct steps of cell migration such as establishment of cell polarity and focal adhesion turnover, however, the molecular mechanism by which RACK1 regulates these processes remains largely unknown. The main aim of this study was to investigate the functional role of RACK1 in cell motility, in particular to identify new effector proteins utilized by the ERK pathway and RACK1 in the regulation of...
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