Functional Characterization of Human ADCK3 and ADCK4, Mitochondrial Atypical Kinases
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AarF domain containing kinases 3 and 4 (ADCK3 and ADCK4, respectively) are paralogous human mitochondrial proteins, co-orthologs of the yeast protein Coq8 and bacterial protein UbiB. These proteins are required in the biosynthesis of coenzyme Q, a lipid-soluble electron carrier well known for its role in energy metabolism, more specifically in the electron transport chain. Patients with mutations in ADCK3 experience an onset of neurological disorders from childhood, including cerebellar ataxia and exercise intolerance. Mutations in ADCK4 cause steroid-resistant nephrotic syndrome, and are also associated with coenzyme Q deficiency. Aside from this minimal characterization at the biochemical level, the precise biological functions of both ADCK3 and ADCK4 remain poorly understood. After extensive screening for soluble recombinant protein expression, N-terminal fusions with maltose-binding protein were found to facilitate the overexpression of human ADCK3 and ADCK4 truncations in Escherichia coli as soluble and biologically active entities. For the first time, our work revealed Mg2+-dependent ATPase activity of ADCK3, providing strong support for the theoretical prediction of these proteins being functional atypical kinases. Subsequent experimentation confirmed protein kinase activity for both ADCK3 and ADCK4. This observed kinase activity was inhibited by the presence of an atypical N-terminal extension – containing an invariant KxGQ motif – found within the kinase domains of these proteins, suggesting an autoinhibitory role for this motif. Additionally, ADCK3 was shown, through radiometric kinase assays, to phosphorylate a peptide corresponding to a potential phosphorylation site in ATP synthase F0 subunit 8, providing insight into a potential role of this atypical kinase family.