ATP Analogs to Study Human Histidine Kinases

The Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Mary Kay Pflum from Wayne State University to determine how the addition of phosphate groups to histidine amino acids in proteins affects their biological functions in mammalian cells. Dr. Pflum is developing analogs of adenosine 5’-triphosphate (ATP) as chemical tools to identify proteins that are phosphorylated at their histidines, in contrast to the more commonly studied phosphorylated amino acids, by kinase enzymes found in human cells. Kinases are associated with a wide variety of cellular events, including cell communication and adaption, and are often overactive in disease states. Thus, the study of kinases and protein phosphorylation impacts the understanding of normal and disease-related biology. This NSF award also encourages a future generation of scientists by exposing middle and high school students to protein structure and function through hands-on outreach activities at local schools. A toolkit of ATP analogs was developed for kinase-catalyzed labeling of protein substrates, which focused in prior work by the PI on serine, threonine, and tyrosine phosphorylation. Given the paucity of chemical tools to study phosphohistidine-mediated mammalian cell biology, the overall goal of this NSF award is to apply kinase-catalyzed labeling with ATP analogs to investigate histidine phosphorylation. Kinase-catalyzed labeling was established with the two known human histidine kinases, NME1 and NME2, using an ATP-biotin analog. Building on kinase-catalyzed labeling, this award focuses on developing several novel chemical tools to study phosphohistidine-containing proteins and substrates for the NME1 and NME2 kinases. Using these chemical tools, critical evidence will be generated to show how histidine phosphorylation influences biological events in mammalian systems. These studies represent essential steps towards our long-term objective to characterize phosphohistidine and histidine kinases in mammalian cellular processes, which is challenging or impossible with available technologies. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.