Structural and functional studies of the Csk and Src family protein tyrosine kinases
Signaling networks are vital to the proper functioning and survival of the cell and the organism. Src family protein tyrosine kinases (SFKs) are involved in a variety of signal transduction events. Activation and over-expression of SFKs lead to development of a variety of human carcinomas. Regulatory mechanisms of Src family members in the cell are not fully understood. ^ One of the known negative regulatory mechanisms for Src is phosphorylation of the C-terminal tyrosine residue by of the Csk family kinases. The Csk family has two members: Csk and Csk homologous kinase (Chk). The presence of two homologous PTKs raises important questions of redundancy and specificity. In Chapter 2 of this dissertation, we expressed, purified and characterized the catalytic properties of Chk. This study showed that Chk and Csk had overall similar catalytic properties, but differed in response to the PP2 inhibitor. ^ Since Csk and Chk are similar in their catalytic function, in Chapter 3, we compared the binding properties of Csk, Chk, and Src SH2 domains and assessed the structural basis for the functional divergence. This study demonstrated a striking functional difference between the Csk and Chk SH2 domains and revealed functional similarities between the Chk and Src SH2 domains. Our data suggested that a single residue in the βD3 position was responsible for the functional differences among the three SH2 domains and revealed a new structure-function relationship for the SH2 domains. ^ Csk phosphorylation of Tyr527 of c-Src triggers the association of the Src SH2 domain with the C-terminal tail and inactivation of the kinase domain. Chapter 4 investigated the intramolecular forces that stabilized inactive conformation of c-Src. We determined that the SH2-tail interaction was not greatly dependent on the affinity between the SH2 domain and the phosphorylated C-terminal tail, but was largely determined by a conformational enhancement. We further demonstrated that side chain of the conserved Trp 260 was a key part of the structural basis for the conformational contribution. These findings suggested that conformational contribution is the predominant force that governs c-Src regulation. ^
Biology, Molecular|Biology, Cell|Chemistry, Biochemistry
"Structural and functional studies of the Csk and Src family protein tyrosine kinases"
Dissertations and Master's Theses (Campus Access).