Access Type

Open Access Dissertation

Date of Award

January 2017

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Mary Kay H. Pflum

Abstract

Kinase-catalyzed phosphorylation plays an important role in cell physiology by regulating a myriad of cellular functions. Thus aberrant kinase activity is implicated in various diseases. Methods are needed to discover kinase substrates and map signaling pathways to explore biology and to help drug discovery. A few techniques are currently available to discover kinase substrate and map cell signaling. However, to augment kinase substrate discovery approaches, it is essential to develop alternative techniques. Pflum has recently discovered cosubstrate promiscuity of protein kinases with gamma-modified ATP analogs. Here, kinase-catalyzed biotinylation with ATP-biotin was used to develop novel tools to discover kinase substrates and to explore kinase signaling cascades.

Initially, a characterization of the generality of kinase-catalyzed biotinylation was performed using 25 different protein kinases and substrates as a collaborative project. Experimental results concluded that all tested protein kinases were capable of transferring a biotin tag to protein substrates, confirming the generality of kinase-catalyzed biotinylation.

Next, kinase-catalyzed biotinylation was applied to develop a substrate-discovery method, entitled, K-BILDS (Kinase-catalyzed biotinylation with inactivated lysates for discovery of substrates) to identify substrates of an interested kinase. Here, kinase inactivated cell lysates were used as the pool of cellular proteins to identify substrates of one particular kinase. As a proof-of-concept, K-BILDS was initially applied to PKA, which resulted in successful identification of ~200 candidate PKA substrates with a ~20% coverage of previously known hits. An interactome analysis and in vitro kinase assay was also performed to validate K-BILDS as a successful tool to discover substrates.

Kinase-catalyzed biotinylation was further applied in to map a cell signaling network. Here, a screen entitled, K-BMAPS (kinase-catalyzed biotinylation to map signaling) was developed by initially applying to EGF-treated cell lysates to map EGFR pathway-related phosphoproteins. K-BMAPS with EGF-treated lysates successfully discovered many EGFR pathway-related phosphoproteins, confirming the ability of K-BMAPS to map a cell signaling network. Thorough analysis established that K-BMAPS detected late and continuous effects of a signaling network, validating the screen to monitor kinase signaling cascades.

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