|
|
Non-traditional roles of formins besides actin nucleation
Metlička, Jáchym ; Cvrčková, Fatima (advisor) ; Opatrný, Zdeněk (referee)
Formin homology 2 (FH2) domaincontaining proteins (formins) have, since their discovery in 1990, been observed in all analyzed species of eukaryotic kingdoms. Our knowledge of structure and function of the defining FH2 domain has greatly increased over the last couple of years. Its function in nucleation, polymerization and processive capping of actin filaments designates formin protein family an important cytoskeletonremodelling factor. But FH2 domain is just one part of the puzzle additional optional conserved peptide structures surrounding it, as well as concrete variation of the FH2 domain itself, greatly influence the functional properties and cellular localization of the resultant formin protein. Formins have been implicated in variety of cellular processes, which often (but not always) involve the cytoskeleton e.g. Factin network management, crosstalk of Factin filaments and microtubules or plasma membrane. They also partake in processes integral to cell division, function in conserved signalling pathways and much more. This thesis explains the structure and function of FH2 and FH1 domains, outlines the main formin phylogenetic clades in multicellular eukaryotes and reviews various roles that formins fulfill or are thought to fulfill. Such goal, however, is very bold and (considering the...
|
|
|
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...
|
|
|
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...
|
|
|
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...
|
|
|
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...
|
|
|
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...
|
|
|
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...
|
|
|
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...
|
|
|
The effect of Crk SH3domain inhibition in invasiveness of cells
Tomášová, Lea ; Rösel, Daniel (advisor) ; Vomastek, Tomáš (referee)
Protooncogene Crk was found to be upregulated in tumours with aggressive and invasive potential. The adaptor protein Crk has an important role in cell signaling: it integrates signals from activated integrins and growth factors receptors via its SH2 domain and transmits the signal to its SH3 domain binding partners that activate the small GTPases Rac1, Rap1 and Ras. This leads to regulation of cell migration, proliferation and survival. The aim of this thesis project was to inhibit the Crk dependent signaling by a competitive inhibition of the Crk SH3 domain, using a high affinity CrkSH3 binding peptoid. Binding of the inhibitor to the Crk SH3 domain prevents binding of cellular Crk SH3 interaction partners and the corresponding signal transmission is impaired. In this thesis project the effect of the Crk SH3 inhibition on the invasiveness of cancer cells was analyzed. The observed inhibitory effect on cell invasion as well as on anchorage independent growth provides a proof of therapeutical relevance of targeting CrkSH3N domain by peptoide-based inhibitors. Powered by TCPDF (www.tcpdf.org)
|
|
|
Biological relevance of the tyrosine 90 phosphorylation in SH3 domain Src kinase
Koudelková, Lenka ; Brábek, Jan (advisor) ; Brdička, Tomáš (referee)
Kinase Src plays an essential role in signal transduction from activated surface receptors. Src is involved in signal pathways that participate in the control of cell proliferation, differentiation or motility. That is why Src activation undergoes strict and complex regulation. Inactive conformation is maintained by intramolecular inhibitory interactions. SH3 domain associates with a polyprolin helix in CD linker whereas SH2 domain binds phosphorylated C-terminal tyrosine 527. Both regulatory domains maintain contacts with the lobes of a kinase domain thereby stabilizing an inactive conformation of the catalytic domain. Transition to an active state is accompanied by a disruption of these inhibitory interactions. Conformation changes are substantially influenced by the phosphorylation status of key tyrosines 416 and 527. Phosphoproteomic analysis revealed new Src tyrosine residue, which can be phosphorylated in vivo. It has been found, that tyrosine works as an additional regulator of Src activity. This is Tyr 90, which forms one of the hydrophobic pockets in the binding surface of Src SH3 domain. Based on the expression of phosphomimic mutant Src 90E in S. pombe or in SYF lineage, it has been observed, that Tyr 90 phosphorylation elevates Src kinase activity. The reason is that the phosphate...
|
guest ::
