Off-campus WSU users: To download campus access dissertations, please use the following link to log into our proxy server with your WSU access ID and password, then click the "Off-campus Download" button below.
Non-WSU users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
Access Type
WSU Access
Date of Award
January 2023
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Chemistry
First Advisor
Mary Kay Pflum
Abstract
Phosphorylation of protein amino acids by kinases regulates mammalian cellular functions, such as signal transduction, growth, and division. Disease development, such as cancer and diabetes, can occur when protein phosphorylation is unregulated. Among human kinases, there are only two known histidine (His) kinases, NME1 and NME2, which are associated with metastatic tumor suppression. However, NME1 and NME2 remain understudied since tools to discover and validate their substrates are lacking, especially for substrates modified to generate heat and acid sensitive phosphohistidine (pHis). To provide new chemical tools to study His kinases in human cell biology, we established several y-phosphate modified ATP analogs as cosubstrates of NME1 and NME2. Building upon ATP-biotin compatibility, the Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K-BILDS) method enabled validation of a known NME2 substrate, KCNN4, and the discovery of seven NME1 and three NME2 substrates that included proteins previously identified as containing pHis. Confirmation studies with K-BILDS supported that PSMA4 and Rho-GDI are NME1 substrates, while UMPS is an NME2 substrate. As a second application of y-phosphate modified ATP derivatives, ATP-propargyl analogs were studied with NME1 and NME2 for reaction efficiency, which was followed by stability assessment of the pHis products generated. Molecular docking was utilized to rationalize the effect of the modified linkage and propargyl groups on human His kinase compatibility. The third ATP analog application utilized ATP-arylazide to establish kinase-catalyzed crosslinking with human His kinases. Crosslinking of recombinant NME1 and in HeLa lysates was assessed, which resulted in formation of the NME1 dimer and higher molecular weight smearing, respectively. Interestingly, the crosslinking was not degraded following heat treatment, which could indicate that the reaction was not solely pHis mediated. Given the paucity of methods to study His kinases, the y-phosphate modified ATP analogs employed here were shown to be useful chemical tools to further characterize the roles of NME1 and NME2 in human cell biology.
Recommended Citation
Gary, Chelsea, "The Study Of Human Histidine Kinases Using Atp Analogs" (2023). Wayne State University Dissertations. 3868.
https://digitalcommons.wayne.edu/oa_dissertations/3868