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The role of p130Cas substrate domain mediated signaling in cancer cell migration, invasiveness and metastasis of cancer cells
Zemanová, Kateřina ; Brábek, Jan (advisor) ; Čáslavský, Josef (referee)
p130Cas (Crk-associated substrate) was first described over 30 years ago as a protein that associates with the v-src and v-crk oncoproteins and undergoes tyrosine phosphorylation. Proteins of the CAS family are an important part of cellular biological processes in normal and pathological situations. The existence of 15 YXXP repetitive motifs is characteristics for substrate domain. p130Cas is an adapter protein that allows interactions between proteins that lead to assembly of multiprotein complexes. The p130Cas protein regulates these multiprotein complexes, which further drive chemotaxis, apoptosis, differentiation and migration. Overproduction of CAS proteins was found in connection with a poor prognosis and an increased incidence of metastases. Also, the elevated expression of proteins of the CAS family is related to resistance to some types of chemotherapeutics.
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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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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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Crosstalk of integrin and mTOR signaling
Teglová, Lucie ; Rösel, Daniel (advisor) ; Libus, Jiří (referee)
iv Abstract Crosstalk of integrin and mTOR signalling is an essential process that monitors cellular interaction with extracellular matrix and transmits these inputs to cell growth signalling. Although adhesion status of the cell monitored by integrin signalling is clearly important for regulation of cellular growth, a little is known about the crosstalk of integrin and mTOR signalling. In this study, we employed two different approaches to describe and elucidate character of this crosstalk. p130Cas is an adaptor protein phosphorylated by Src kinase and focal adhesion kinase upon integrin ligand binding and implicated in cell adhesion, motility and survival in both Src-transformed and untransformed cells. Recently, p130Cas was also described in cellular pathology, mainly by its ability to stimulate cell invasion and metastasis. In this study, we described that p130Cas affects mTOR signalling in Src-transformed cells. Substrate domain of p130Cas was found to be indispensable for this effect and is also responsible for serum-induced activation of mTOR signalling. In addition, we prepared cell lines overexpressing various Rheb protein versions and characterized them in context of mTOR signalling, integrin signalling and cell cycle progression. Interestingly, a cell line overexpressing constitutively active...
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The search for novel interaction partners of SH3 domain of an adaptor protein p130Cas
Gemperle, Jakub ; Rösel, Daniel (advisor) ; Forstová, Jitka (referee)
Protein p130Cas is the major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. P130Cas plays an important role in invasiveness and metastasis of Src-transformed cells. In breast cancer patients, high p130Cas levels are associated with higher recurrence of disease, poor response to tamoxifen treatment and lower overall survival. In non-transformed cells, after the stimulation of integrins, protein p130Cas is phosphorylated in substrate domain affecting cell migration and cytoskeletal dynamics. For this signalling is the SH3 domain of p130Cas indispensable. In this thesis, was for the first time using the Phage display method analysed and subsequently characterized the binding motif of SH3 domain of p130Cas. Based on this high-affinity motif [AP]-P-[APMS]-K-P-[LPST]-[LR]- [LPST], we predicted new interaction partners of protein p130Cas and subsequently confirmed the interaction with the Ser/Thr kinase PKN3. This kinase colocalizes with p130Cas in the nucleus and perinuclear region and could phosphorylate p130Cas. In this thesis, we also analysed the effect of phosphomimicking mutation of tyrosine from sequence ALYD, which is conserved in the sequence of SH3 domains, on ability of these domains to bind ligands. This mutation reduced binding by about 3 orders of...
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Analyzing the role of the p130Cas SH3 domain in p130Cas-mediated signaling
Gemperle, Jakub ; Rösel, Daniel (advisor) ; Vomastek, Tomáš (referee) ; Truksa, Jaroslav (referee)
The adaptor protein p130Cas (CAS, BCAR1) represents a nodal signaling platform for integrin and growth factor receptor signaling, and influences normal development and tissue homeostasis. Its altered expression drives many pathological conditions including tumor growth, metastasis and drug resistance in many cancer types. How p130Cas contributes to many of these pathologies is still poorly understood. Therefore, the overall aim of my PhD work was to provide new insights to p130Cas signaling and its regulation. The SH3 domain is indispensable for p130Cas signaling, but the ligand binding characteristics of the p130Cas SH3 domain, and the structural determinants of its regulation were not well understood. To be able to study various aspects of p130Cas signaling we identified an atypical binding motif in p130Cas SH3 domain by establishing collaborations with Dr Veverka (Structural biology) and Dr Lepšík (Computational biochemistry; Academy of Sciences, CZ) which gave new insight into this binding interface. Through these collaborations I generated chimeras of p130Cas SH3 domain with its ligands for structural NMR analysis and learned how to visualize and analyze structures. Furthermore, my work expanded our knowledge of p130Cas SH3 ligand binding regulation and led to a novel model of Src-p130Cas- FAK...
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The role of p130CAS in mechanosensing
Braniš, Jaroslav ; Brábek, Jan (advisor) ; Gregor, Martin (referee)
Sensing mechanical stimuli by proteins and their conversion into biochemical signals in a mechanotransduction is the recently discussed topic in cell biology. The main molecules that cells use to sense mechanical stimuli, are membrane proteins that transmit the mechanical stimulus into the cell. In the cell are found protein complexes, which transmit the signal further. One of the most important protein that has the ability to change the mechanical stimuli to biochemical signals and transmit them to other protein is p130Cas. This work deals with the ability of p130Cas to sense mechanical stimuli and transmit them to other proteins and signaling pathways that regulate cellular response depending on the mechanical stress. Key words: p130Cas, p130Cas/Crk komplex, mechanical stress
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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 substrate domain mediated signaling in cancer cell migration, invasiveness and metastasis of cancer cells
Zemanová, Kateřina ; Brábek, Jan (advisor) ; Čáslavský, Josef (referee)
p130Cas (Crk-associated substrate) was first described over 30 years ago as a protein that associates with the v-src and v-crk oncoproteins and undergoes tyrosine phosphorylation. Proteins of the CAS family are an important part of cellular biological processes in normal and pathological situations. The existence of 15 YXXP repetitive motifs is characteristics for substrate domain. p130Cas is an adapter protein that allows interactions between proteins that lead to assembly of multiprotein complexes. The p130Cas protein regulates these multiprotein complexes, which further drive chemotaxis, apoptosis, differentiation and migration. Overproduction of CAS proteins was found in connection with a poor prognosis and an increased incidence of metastases. Also, the elevated expression of proteins of the CAS family is related to resistance to some types of chemotherapeutics.
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