Methods for Identifying Substrates of Atypical Mitochondrial Kinases

Biochemistry

Barber, GrantPagliarini, David ;

University of Wisconsin

关键词: Biochemistry;   

Others  :  https://minds.wisconsin.edu/bitstream/handle/1793/67917/2012_Barber.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: University of Wisconsin

【 摘 要 】

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 itsmembers 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.