Kinase enzymes are responsible for the reversible phosphorylation of numerous proteins involved in signal transduction cascade pathways, which regulate key cellular processes such as growth and metabolic activity. Kinases function as transferases by adding the gamma-phosphoryl group from Adenosine 5’-triphosphate (ATP) onto serine, threonine, or tyrosine residues of protein substrates. The Pflum lab discovered that strategic modification of the gamma-phosphate group of ATP generated kinase-compatible ATP analog cosubstrates capable of labeling kinase protein substrate(s). Based on successful kinase-catalyzed labeling with these gamma-phosphate modified ATP analogs, kinase-catalyzed crosslinking of kinase and protein substrate was developed to bridge the gap in kinase-substrate pair identification. However, the method has been limited to use of photocrosslinking ATP-Aryl Azide, which requires use of UV light that is harmful to cellular proteins and DNA. In addition, UV light exposure creates a highly unstable nitrene group on ATP-Aryl Azide that reacts with many surrounding proteins and leads to incorrect kinase-substrate pair identification. To avoid these problems, new ATP analogs have been synthesized and tested by the Pflum lab for use in kinase-catalyzed crosslinking. In this project, new ATP analogs from the Pflum lab were characterized via kinase kinetics assays and gel analysis following kinase-catalyzed crosslinking. Through development of new kinase compatible ATP analogs, kinase-substrate pairs can be more accurately identified for a better understanding of kinase-dependent signal transduction pathways.

Development and characterization of novel ATP analogs for studying protein phosphorylation and kinase enzymes

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