AMP-activated protein kinase (AMPK) is the master regulator of eukaryotic metabolism and energetics, activating catabolic pathways to generate ATP and inhibiting anabolic to prevent rapid expenditure of ATP. It has been determined that AMPK targets the phosphagen kinase (PK) family enzyme creatine kinase in humans and rabbits. This knowledge sparks interest in characterizing the evolutionary origins of the AMPK-PK regulatory relationship across species. Last year Andrew Greene began preliminary research into this topic and argued that PKs must exhibit a flexible N-terminal region to be targeted by AMPK. Additionally, he claimed that F44, an arginine kinase in the invertebrate roundworm Caenorhabditis elegans, is targeted by AMPK. While Greene provides a convincing argument and fascinating conclusions in his study, the results from this thesis call the validity of his work into question. When aiming to continue this investigation, utilizing the same in vitro thiophosphorylation assay to identify probable AMPK targets, it quickly became apparent that the assay was not specifically phosphorylating PK proteins. The trouble in reproducing Greene’s results primed an investigation into the biochemical reaction of the assay used to screen the AMPK substrates. We hypothesize that under these assay conditions, the upstream kinase activator of AMPK (liver kinase β-1) autophosphorylates, preventing it from phosphorylating the catalytic subunit in AMPK and thereby halting the kinase cascade and preventing any potential PK phosphorylation. This study provides a thorough investigation in to the mechanistic intricacies of the thiophosphorylation assay with the intentions to optimize assay protocol and improve understanding of the AMPK-PK regulatory relationship.
Biochemistry and Molecular Biology
Boufford, Camille K., "Mechanistic Investigation into the Liver Kinase β-1 – AMP-Activated Protein Kinase – Phosphagen Kinase Phosphorylation Cascade" (2017). Senior Independent Study Theses. Paper 7676.
Bachelor of Arts
Senior Independent Study Thesis
© Copyright 2017 Camille K. Boufford