Cancer, a disease characterized by dangerous and uncontrollable cell growth and division, is often caused by abnormal activity levels of both tumor inducing and tumor suppressing proteins. One protein that has been shown to promote cancer progression when mutated or dysregulated is tyrosine kinase Src, which phosphorylates proteins involved in cell morphology, motility, proliferation, and survival pathways. Previous studies of Src have found two conformational features (the αC-helix and activation loop) that determine whether the enzyme is in its active or inactive state, and different types of kinase inhibitors bind these different conformations of Src. In order to investigate the efficacy of the three most common classes of kinase inhibitors against Src kinase mutants, a high throughput scan of a library of single amino acid Src mutants treated with various kinase inhibitors was performed. This experiment resulted in preliminary relative activity levels of all Src mutants in the library along with how these activity levels changed in the presence of each of the three inhibitors.
This study investigates specific Src mutants’ activity levels and inhibitor sensitivity seen in the high throughput scan through individual validation experiments. Site-directed mutagenesis was used to generate these mutants, and then a functionality assay was used to determine the activity levels of each mutant in comparison to wildtype Src. The relative activity levels in the no inhibitor condition of the four Src mutants used in this study correlated closely with the expected activity levels seen in the high throughput scan. Furthermore, Src mutant V331E demonstrated the same differential resistance to two different kinase inhibitors that was seen in the inhibitor specific high throughput scans. This mutant appeared to be sensitive to one inhibitor and resistant to another in both the high throughput scans and the individual validation experiment. Mutation V331E’s proximity to the conformation determining αC-helix may explain this differential resistance. Further studies are necessary to investigate the precise structural changes in Src that occur as a result of this mutation in order to understand the differential resistance seen in this mutant.
| Identifer | oai:union.ndltd.org:CLAREMONT/oai:scholarship.claremont.edu:cmc_theses-3160 |
| Date | 01 January 2019 |
| Creators | Ketavarapu, Gayatri |
| Publisher | Scholarship @ Claremont |
| Source Sets | Claremont Colleges |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | CMC Senior Theses |
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