Date of Award
2005
Degree Type
Dissertation
First Advisor
Gongqin Sun
Abstract
Protein phosphorylation, enzymatic transfer of γ-phosphate of adenosine triphosphate to a hydroxyl group of a recipient amino acid residue such as serine, threonine or tyrosine, is a fundamental control mechanism of cell signaling in eukaryotes. Most protein kinases, catalyzing protein phosphorylation, consist of multiple globular domains which play important roles in connecting signal transduction pathways. Due to their critical roles in cell signaling and the correlation between their activity and disease processes of the cell, many protein tyrosine kinases (PTKs) are targets for inhibitor development. For the purposes of understanding their mechanisms in signal transduction and developing PTK inhibitors for therapeutic applications, establishing the function-structure relationships of PTKs is an important area of research. Here I describe the biochemical approaches guided by the structural information and the computational methods in order to understand the function-structure relationship of a model PTK, the C-terminal Src Kinase (Csk). Site-directed mutagenesis of tryptophan (Trp) residues revealed that the enhanced and blue-shifted spectrum of intrinsic Trp fluorescence of Csk was due to the presence of noncovalent interaction force, cation-pi (π) interaction between two amino acid residues, R318 and W352, located in the active site of Csk. The disruption of such interaction by site-specific mutation has significant effect on Csk catalyzing its substrate and binding of protein substrate, Src. The mechanisms that determine the substrate specificity of PTKs have been elusive since the discovery of c-Src, the proto-tyrosine kinase. We demonstrated that Csk phosphorylated Src family kinases specifically through the docking-based substrate recognition. The α helix D and connected loop of Csk and α helix J of Src were determined as the docking site. Mutations that abolished the Csk-Src docking interaction caused reduced phosphorylation of Src, insensitivity to Csk regulation, and impaired physical binding. Here we proposed novel paradigm of substrate recognition mechanism of PTKs, the docking-interaction based substrate recognition. This molecular level understanding of substrate specificity will not only provide a model for understanding substrate specificity in other PTKs, but also help to build a knowledge base for PTK inhibitor design. In the future, it may be possible to design a docking-based kinase inhibitors.
Recommended Citation
Lee, Sungsoo, "Functional and structural study of the protein tyrosine kinase Csk, as a model system" (2005). Open Access Dissertations. Paper 2046.
https://digitalcommons.uri.edu/oa_diss/2046
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