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Adaptor domains in signalling proteins: phosphorylation analysis and a role in mechanosensing
Tatárová, Zuzana ; Novotný, Marian (advisor) ; Doležal, Pavel (referee)
P130Cas (Crk-associated substrate, CAS) is a multiadaptor protein important in integrin signalling where it positively regulates cell motility, invasion, proliferation and survival. CAS lacks enzymatic activity, but its binding to other signalling proteins could lead to the change of phosphorylation status of its substrate domain, which is the main mode, through which CAS takes part in regulating cell behavior. Local tensions in focal adhesions lead to an extension of CAS substrate domain, leaving phosphorylation sites more accessible for kinases, which subsequently leads to an increased CAS substrate domain phosphorylation. The CAS anchorage in focal adhesions is mediated by its SH3 domain, probably through the interactions with FAK, and also by C-terminal domain, where interaction partners are not known. The aim of my project is to find out, which proteins mediate the CAS anchorage to the focal adhesions. The elucidation of CAS anchorage to focal adhesions will contribute to the understanding of mechanosensory function of CAS. Experimental data suggest that tyrosine phosphorylation of the CAS SH3 domain plays an important role in the regulation of its binding properties. Another goal of my diploma project was to analyze the significance of tyrosine phosphorylation within SH3 domain and other...
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The biological importance of CAS SH3 domain tyrosine phosphorylation
Janoštiak, Radoslav ; Brábek, Jan (advisor) ; Dvořák, Michal (referee)
Protein CAS is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes. It is a multidomain adaptor protein, which serves as a scaffold for assembly of signalling complexes which are important for migration and invasiveness of Src-transformed cells. A novel phosphorylation site in N-terminal SH3 domain was identified - tyrosine 12 located on binding surface of CAS SH3 domain. To study biological importance of tyrosine 12 phosphorylation, non-phosphorylable (Y12F) and phosphomimicking ( Y12E) mutant of CAS were prepared. We found that phosphomimicking mutation Y12E leads to decreased interaction of CAS SH domain with kinase FAK a phosphatase PTP-PEST and also reduce tyrosine phosphorylation of FAK. Using GFP-tagged CAS protein, we show that Y12E mutation caused delocalization of CAS from focal adhesion but has no effect on localization of CAS to podosome-type adhesion. Non-phosphorylable mutation Y12F cause hyperphosphorylation of CAS substrate domain and decrease turnover of focal adhesion and associated cell migration of mouse embryonal fibroblasts (MEFs) independent to integrin singalling. Analogically to migration, CAS Y12F decrease invasiveness of Src-transformed MEF. The results of this diploma thesis show that phosphorylation of Tyr12 in CAS SH3 domain is...
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New regulatory mechanisms of microtubule nucleation
Černohorská, Markéta ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee) ; Hašek, Jiří (referee)
MT nucleation from γ-tubulin complexes, located at centrosome, is an essential step in the formation of MT cytoskeleton. In mammalian cells, -tubulin is encoded by two genes. We functionally characterized two γ-tubulin proteins and have found that both are functionally equivalent. γ-Tubulin 2 is able to substitute for γ-tubulin 1 in MT nucleation. However, we revealed that unlike TUBG1, TUBG2 expression is downregulated in mouse preimplantation development. Mast cells represent effectors of the allergy reaction. Their activation by antigen induces number of cellular processes such as degranulation, proliferation and cytoskeleton rearrangements. The regulatory mechanisms of MT reorganization during mast cell activation are unknown. We identified new signaling proteins, GIT1 and PIX that interact with - tubulin. Depletion of GIT1 or PIX leads to changes in MT nucleation. GIT1 is phosphorylated on tyrosine and associates with γ-tubulin in a Ca2+ -dependent manner. Our data suggested a novel signaling pathway for MT rearrangement in mast cells where tyrosine kinase-activated GIT1 and βPIX work in concert with Ca2+ signaling to regulate MT nucleation. We tested the capability of GIT1 and PIX to influence -tubulin function in more cell types. We found out that GIT1/βPIX signaling proteins together...
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The influence of estrogens on mouse sperm capacitation and acrosome reaction in vitro
Tejnická, Magda ; Komrsková, Kateřina (advisor) ; Linhart, Otomar (referee)
There are an increasing amount of compounds in the environment that can have a negative effect on reproductive parameters in both male and female organism. There has been a worldwide decline of sperm quality during past decades and this fact lead to an increase of unnatural ways of conception through assisted reproduction techniques in the specialised centres. Natural estrogens are one of these compounds and they get into waste water after being excluded from the body by the urine. They get back into the human body from drinking water or from the food, and they can interfere with function of endogenous hormones in very low concentrations. For these reasons it is up to date to deal with the influence of these compounds on mammalian sperm. For many years, estrogens have been considered typically female sex hormones. It is now certain that they are also very important in the regulation of male reproduction. Endogenous estrogens in mammalian males are an important part of the endocrine system. Estrogens play an important role in the development of germ cells, spermatogenesis and processes leading to successful egg fertilization such as a capacitation or acrosomal reaction. Tyrosine phosphorylation is one of the essential steps for the properly ongoing process of capacitation in sperm followed by a...
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Adaptor domains in signalling proteins: phosphorylation analysis and a role in mechanosensing
Tatárová, Zuzana ; Novotný, Marian (advisor) ; Doležal, Pavel (referee)
P130Cas (Crk-associated substrate, CAS) is a multiadaptor protein important in integrin signalling where it positively regulates cell motility, invasion, proliferation and survival. CAS lacks enzymatic activity, but its binding to other signalling proteins could lead to the change of phosphorylation status of its substrate domain, which is the main mode, through which CAS takes part in regulating cell behavior. Local tensions in focal adhesions lead to an extension of CAS substrate domain, leaving phosphorylation sites more accessible for kinases, which subsequently leads to an increased CAS substrate domain phosphorylation. The CAS anchorage in focal adhesions is mediated by its SH3 domain, probably through the interactions with FAK, and also by C-terminal domain, where interaction partners are not known. The aim of my project is to find out, which proteins mediate the CAS anchorage to the focal adhesions. The elucidation of CAS anchorage to focal adhesions will contribute to the understanding of mechanosensory function of CAS. Experimental data suggest that tyrosine phosphorylation of the CAS SH3 domain plays an important role in the regulation of its binding properties. Another goal of my diploma project was to analyze the significance of tyrosine phosphorylation within SH3 domain and other...
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|
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The biological importance of CAS SH3 domain tyrosine phosphorylation
Janoštiak, Radoslav ; Brábek, Jan (advisor) ; Dvořák, Michal (referee)
Protein CAS is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes. It is a multidomain adaptor protein, which serves as a scaffold for assembly of signalling complexes which are important for migration and invasiveness of Src-transformed cells. A novel phosphorylation site in N-terminal SH3 domain was identified - tyrosine 12 located on binding surface of CAS SH3 domain. To study biological importance of tyrosine 12 phosphorylation, non-phosphorylable (Y12F) and phosphomimicking ( Y12E) mutant of CAS were prepared. We found that phosphomimicking mutation Y12E leads to decreased interaction of CAS SH domain with kinase FAK a phosphatase PTP-PEST and also reduce tyrosine phosphorylation of FAK. Using GFP-tagged CAS protein, we show that Y12E mutation caused delocalization of CAS from focal adhesion but has no effect on localization of CAS to podosome-type adhesion. Non-phosphorylable mutation Y12F cause hyperphosphorylation of CAS substrate domain and decrease turnover of focal adhesion and associated cell migration of mouse embryonal fibroblasts (MEFs) independent to integrin singalling. Analogically to migration, CAS Y12F decrease invasiveness of Src-transformed MEF. The results of this diploma thesis show that phosphorylation of Tyr12 in CAS SH3 domain is...
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