Probing the regulatory mechanisms of protein tyrosine kinases, using c-terminal Src kinase (Csk) as a model system
Protein tyrosine kinases (PTKs) are important signal transducers. Most PTKs have a catalytic domain, which carries out catalysis, and regulatory domains, which regulate their activity. Autophosphorylation, the Tyr phosphorylation in the activation loop, is also a key regulatory mechanism for most PTKs. In this study, I probed the regulatory mechanisms of PTKs using Csk as a model system. ^ Csk has two regulatory domains, SH3 and SH2, and a catalytic domain. In this study, I probed how the regulatory domains modulate Csk activity. First, the regulatory domains were required for Csk full activity, yet not involved in the recognition of Src, the physiological substrate of Csk. Second, our data indicated that Csk regulatory domains modulated multiple substructures in the catalytic domain. Third, two key interactions between the catalytic domain and the regulatory domains were identified. ^ Another mode of Csk regulation was through the SH2 domain ligands. In this study, I probed how the SH2 domain activates Csk catalytic domain by binding to a ligand. Six residues, three from the catalytic domain and three from the SH2 domain, were identified to be important in this domain-domain communication. Among the six, three play roles in suppressing Csk activity, suggesting that the SH2 domain suppresses Csk activity, and its binding to the ligands relieves this suppression. ^ Different from most PTKs, Csk has no Tyr in its activation loop and is not regulated by autophosphorylation. In this study, I probed the roles of the Csk activation loop. First, specific residues in this loop were not important to Src phosphorylation. Second, some loop mutants exhibited significantly higher activity toward Src than toward artificial substrates. Third, introduction of a thrombin cleavage site to the loop resulted in significant activity loss toward both substrates, but digestion with thrombin resulted in full activity recovery, implying an inhibitory role of this loop. ^ In summary, I probed the regulation of Csk by the presence of the regulatory domains, by the SH2 domain ligands, and by the activation loop. This study will help in understanding the regulation of other PTKs. ^
"Probing the regulatory mechanisms of protein tyrosine kinases, using c-terminal Src kinase (Csk) as a model system"
Dissertations and Master's Theses (Campus Access).