Date of Award
2009
Degree Type
Dissertation
First Advisor
Gongqin Sun
Abstract
The Src and FGFR family of protein tyrosine kinases (PTKs) are key mammalian enzymes in signal transduction. Regulating their enzymatic activity is imperative and disrupting this regulation can lead to devastating outcomes, including cancer. Our primary focus is to dissect the structure-function relationship that guides the regulation of PTKs. In this study we used site directed mutageneis, bacterial expression, and kinetic analysis to investigate how Src and FGFR PTKS is regulated by Reactive Oxygen Species (ROS) and autophosphorylation. Accumulating evidence suggests that protein tyrosine phosphorylation-based pathways are under the regulation of Reactive Oxygen Species (ROS). Although protein tyrosine phosphatases are firmly established to be reversibly regulated by ROS, it is not clear if PTKs are also directly regulated by reduction/oxidation (redox). In this study we report the Src and FGFR family of PTKs are reversibly inactivated by oxidation of a specific cysteine residue in the highly conserved Glycine rich loop, C277 in Src and C488 in FGFR1. This cysteine residue is uniquely conserved in only 8 of more than 90 PTKs in the human kinome, seven of which are in the Src and FGFR families. Csk, which lacks a cysteine at the equivalent position, was able to be engineered to become redox sensitive by single amino acid substitution. This is the first line of evidence to establish a direct mechanism by which PTKs are regulated by ROS. Autophosphorylation of the activation loop stimulates the catalytic activity of many protein kinases and is strongly associated with their oncogenic properties. Despite the importance of Autophosphorylation it is unclear how autophosphorylation is regulated. In this study we reveal that autophosphorylation of Wt-FGFR2 is highly suppressed, precluding its activation by autophosphorylation. The suppression was determined to be mediated by the identity of R678, in which R678E dramatically activated the rate of autophosphorylation and directly correlates to a 20 to 30 fold autoactivation of FGFR2 Furthermore these mutants which release suppression of FGFR2 autophosphorylation are not all that different in kinase activity prior to autoactivation, illustrating that kinase activity and autophosphorylation are independent of one another.
Recommended Citation
Kemble, David J., "A biochemical study on the regulation of the Src and FGFR family of protein tyrosine kinases" (2009). Open Access Dissertations. Paper 2288.
https://digitalcommons.uri.edu/oa_diss/2288
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