Peptide-based biosensors and light-activatable proteins: tools for studying cell signaling and developing cancer diagnosis Public Deposited

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  • March 22, 2019
  • Xu, Weichen
    • Affiliation: Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry
  • Protein kinases play central roles in almost all aspects of normal and abnormal cell functions. Therefore, methods that provide sensitive, selective and controllable measurement of kinase activity should prove useful for studying cell signaling events. Moreover, easy to operate, generalizable strategies are in high demand for disease diagnosis. This dissertation described the development and application of peptide-based kinase biosensors and strategies to control enzyme activities by light. First, we developed a highly sensitive peptide-based biosensor that employs capillary electrophoresis to measure Src activity in prostate cell lysates as well as in single living cells. Our studies revealed that there is a direct correlation between cell aggressiveness and the fraction of cellular Src that is in the active state. We also determined that pTyr-527 level of Src is a poor indicator of inactive Src. In addition, we have demonstrated that two or more kinases can be simultaneously monitored using this technique. Second, aimed at unraveling the distinct roles of the Src family kinases Fyn and Lck in T cell activation, we developed a highly selective, potent, and light-deactivatable Fyn inhibitor. This inhibitor provides a way of selectively activating Fyn kinase by light. We have also discovered that attaching a profluorescent dye rendered this peptide cell permeable. Third, we described a new strategy for the construction of a profluorescent light-activatable cAMP-dependent protein kinase (PKA) via active site-directed peptide-based affinity labeling. This modified PKA displays minimum catalytic activity and low fluorescence. Light illumination restored enzymatic activity and induced stress fibers lose in living cells. A 6.2-fold fluorescence enhancement was also observed. The strategies developed in these studies provided powerful tools to elucidate the relationship of kinase activity with disease stages and the complex cell signaling networks. With these tools in hand, we are hoping to advance our knowledge of a basic biological phenomenon as well as providing future diagnostic tool for cancer patients.
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  • In Copyright
  • Lawrence, David
  • Doctor of Philosophy
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  • 2012

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