Methods for Identifying Substrates of Atypical Mitochondrial Kinases
Abstract
Mitochondria are chiefly tasked with the production of ATP. Reversible phosphorylation is postulated to be a central regulatory mechanism in mitochondrial biology, and yet the mitochondrial kinases that perform the phosphorylation events are largely uncharacterized[l]. One group of atypical mitochondrial kinases, the ADCKs
(aarF-domain containing kinases), has garnered interest because of the role one of its
members play in human disease. Here, we designed and validated a radiolabeling based approach to identify substrates of atypical mitochondrial kinases. To detect labeled proteins, we optimized SDS-PAGE; to detect labeled lipids, we optimized thin layer chromatography (TLC). By manipulating kinase reaction conditions, we reduced background phosphorylation of proteins and lipids by endogenous kinases. From there, we established a high-throughput, in vitro kinase assay to validate kinase-substrate pairs. Our next step is to use this approach to identify substrates of the ADCK family to elucidate their roles in human health and disease.
Subject
Biochemistry
Permanent Link
http://digital.library.wisc.edu/1793/67917Description
24 p.