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Access Type

WSU Access

Date of Award

January 2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Mary Kay H. Pflum

Abstract

The kinase family consist of a large assortment of enzymes that phosphorylate a variety of substrates, including proteins, nucleosides, polynucleotides, lipids, sugars, and other small molecules. The Pflum lab recently found that protein kinases promiscuously accept an ATP analog containing a biotin at the γ-phosphate (ATP-biotin) as a cosubstrate to label the hydroxyl side chains of Ser, Thr and Tyr residues with biotin. The main goal of this new project is to extend the use of kinase-catalyzed biotinylation towards polynucleotides. Crystallographic studies of T4 polynucleotide kinase (T4 PNK) that phosphorylates the 5’-hydroxyl termini of DNA and RNA show a solvent exposed ATP binding site, which could facilitate the binding of γ-phosphate modified ATP to the enzyme. Based on this analysis, we hypothesize that T4 polynucleotide kinase could also transfer a phosphobiotinyl group from ATP-biotin to a single stranded DNA substrate. Preliminary data are consistent with kinase-catalyzed biotinylation. Once validated, kinase-catalyzed biotinylation can be employed as a novel chemical tool to biotinylate the 5’-end of single / double stranded DNA or RNA as an alternative to radioactive or chemical labeling.

Lipids play an important role in cell biology. Lipid kinases phosphorylate a variety of lipid molecules, including phosphatidylinositols, ceramides, sphingosines, and diacylglycerols. To date, our lab has focused entirely on studies of ATP cosubstrate promiscuity on protein kinases. The crystallographic analysis of various lipid kinases shows solvent exposed binding sites for ATP co-substrate as same as the protein kinases, that exhibit co-substrate promiscuity. Based on these facts we hypothesize that lipid kinases would also exhibit ATP co-substrate promiscuity. Therefore, an alternative, chemical tool is proposed to study lipids, based on kinase-catalyzed labeling in understanding lipid based cell signaling.

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