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To be or not to be activated: current view on the regulation of Lck in the proximal T-cell signaling
Valečka, Jan ; Filipp, Dominik (advisor) ; Brdička, Tomáš (referee)
Src-family kinases are critical members of many signalling pathways present in many cell types. Lck, a member of this family of kinases, plays a crucial role in the initiation of T cell activation. An activation of Lck in T cells is one of the first biochemical events detectable after T cell receptor engagement. Kinase activity of Lck is regulated on several levels. Firstly, activity of Lck depends on phosphorylation status of positive and negative regulatory tyrosines that predicate structural conformation and kinase activity. Several kinases and phosphatases targeting Lck regulatory tyrosine residues have been already characterized. Another level of complexity in the regulation of Lck activity is mediated through interactions with adaptor proteins. A critical role in the regulation of Lck activity plays the cellular microenvironment, especially membrane microdomains called Lipid rafts. For a long time, it was assumed that ligand recognition by TCR triggers Lck activation what in turn initiates the downstream signalling. However this paradigm has been challenged as recent data point to the preexistence of a large fraction of Lck in activated state prior to TCR triggering and remains so during the first 2 - 5 minutes after activation. This novel finding poses new questions about the mechanisms that (i)...
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Identification of a new mechanism of Lck regulation via its C-terminal sequence
Valečka, Jan ; Filipp, Dominik (advisor) ; Vomastek, Tomáš (referee)
T-cell activation is a complex process crucial for a proper function of immune system. It has been extensively studied and its main features are well understood. However, some of the events involved in T-cell signalling are still unclear. After T-cell receptor stimulation, Src-family kinase Lck drives the initiation of signalling by tyrosine phosphorylation. Phosphorylation of several downstream targets is dependent on the redistribution of Lck to the different compartment of the plasma membrane, called lipid rafts. In lipid rafts, active Lck is juxtaposed and activates raft-resident substrates which then trigger downstream signalling. The critical in this process is the mechanism of Lck translocation to lipid rafts which has not been studied so far and represents the topic of great academic and clinical interests. Previously, we identified the adaptor protein RACK1 as a candidate protein mediating the redistribution of Lck to lipid rafts by linking it to the microtubular network. In this thesis, we analysed the structural features and functional role of RACK1 in its interaction with Lck. We show here, using the SYF cell lines expressing the wild type and various mutated forms of Lck, that intact SH3 or SH2 domains of Lck are required for an effective RACK1-Lck complex formation. We also documented...
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To be or not to be activated: current view on the regulation of Lck in the proximal T-cell signaling
Valečka, Jan ; Filipp, Dominik (advisor) ; Brdička, Tomáš (referee)
Src-family kinases are critical members of many signalling pathways present in many cell types. Lck, a member of this family of kinases, plays a crucial role in the initiation of T cell activation. An activation of Lck in T cells is one of the first biochemical events detectable after T cell receptor engagement. Kinase activity of Lck is regulated on several levels. Firstly, activity of Lck depends on phosphorylation status of positive and negative regulatory tyrosines that predicate structural conformation and kinase activity. Several kinases and phosphatases targeting Lck regulatory tyrosine residues have been already characterized. Another level of complexity in the regulation of Lck activity is mediated through interactions with adaptor proteins. A critical role in the regulation of Lck activity plays the cellular microenvironment, especially membrane microdomains called Lipid rafts. For a long time, it was assumed that ligand recognition by TCR triggers Lck activation what in turn initiates the downstream signalling. However this paradigm has been challenged as recent data point to the preexistence of a large fraction of Lck in activated state prior to TCR triggering and remains so during the first 2 - 5 minutes after activation. This novel finding poses new questions about the mechanisms that (i)...
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