Access Type

Open Access Dissertation

Date of Award

January 2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Mary Kay H. Pflum

Abstract

Kinase-catalyzed protein phosphorylation is one of the most important post-translational modifications that controls cascades of biochemical reactions. Irregularities in phosphorylation result in many diseases, such as diabetes mellitus, Parkinsons, and cancer. The development of new methods to monitor kinase-catalyzed phosphorylation is needed to decipher details of normal and diseased cell signaling. The Pflum lab recently developed several -modified ATP analogs to study kinase catalyzed phosphorylation reactions. The -modified ATP analogs have different tags, such as biotin for substrate labeling or aryl-azide for kinase substrates identification. Unfortunately, use of -modified ATP analogs was limited to in vitro studies due to the cell impermeability of ATP analogs. Here, we report the first cell permeable ATP analog compatible with kinase-catalyzed labeling. Cell permeable ATP-biotin showed in vitro protein labeling similar to the previously reported ATP-biotin. Importantly, biotin labeling of kinase substrates in living cells was also observed. Also, we report an alternative method to permeabilize ATP analogs in cases where synthesis of cell permeable ATP analog is not feasible. Permeabilizing ATP analogs will aid in monitoring kinase-catalyzed phosphorylation in living cells, which will enhance studies on cell signaling cascades and disease formation. Furthermore, we developed new crosslinking ATP analogs to identify substrates to kinases. We report affinity-based crosslinking ATP analogs, ATP-acrylamides. ATP-acrylamides crosslinked cysteine containing kinases to their substrates through specific proximal cysteine residues on kinases. ATP-acrylamides will solve problems accompanied with the previously reported ATP-Ar-azide that suffer from nonspecific crosslinking due to the very reactive crosslinking azide group. ATP-acrylamides will provide the details of cell signaling of specific kinases, which is supporting biomedical studies. Lastly, we are studying the effect of different bonds between the -phosphate and the attached group on kinase cosubstrate promiscuity phenomenon. This study revealed the electronic effect of several atoms on the ability of ATP-analogs to act as kinase-cosubstrates. Understanding the factors governing the ability of ATP analogs to act as cosubstrate will lead to development of more suitable ATP analogs for kinase catalyzed phosphorylation studies.

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