Loss of either PINK1 or PRKN causes an early onset Parkinson’s disease (PD) phenotype. Functionally, PINK1 and PRKN work together to mediate stress-activated mitochondrial quality control. Upon mitochondrial damage, PINK1, a ubiquitin kinase and PRKN, a ubiquitin ligase, decorate damaged organelles with phosphorylated ubiquitin for sequestration and degradation in lysosomes, a process known as mitophagy. While several genetic mutations are established to result in loss of mitophagy function, many others have not been extensively characterized and are of unknown significance. Here, we analyzed a set of twenty variants, ten in each gene, focusing on understudied variants mostly from the Parkinson’s progressive marker initiative, with sensitive assays to define potential functional deficits. Our results nominate specific rare genetic PINK1 and PRKN variants that cause loss of enzymatic function in line with a potential causative role for PD. Additionally, we identify several variants with intermediate phenotypes and follow up on two of them by gene editing midbrain-derived neuronal precursor cells. Thereof derived isogenic neurons show a stability defect of the rare PINK1 D525N mutation, while the common PINK1 Q115L substitution results in reduced kinase activity. Our strategy to analyze variants with sensitive functional readouts will help aid diagnostics and disease treatment in line with current genomic and therapeutic advances.

The ADP-ribosyltransferase tankyrase (with two paralogues, TNKS and TNKS2) plays pivotal roles in diverse cellular processes that encompass signal transduction, including Wnt/β-catenin, Hippo and toll-like receptor (TLR) signalling, mitotic spindle assembly, glucose homeostasis and telomere maintenance, among many other functions. Tankyrase recruits its effectors (substrates and binders) via a degenerate tankyrase-binding motif (TBM) and exerts its activities by subsequent substrate ADP-ribosylation and/or scaffolding. Variants of the TBM, found in diverse proteins, engage the ankyrin repeat cluster (ARC) domains of tankyrase. Yet, whether effector recruitment to tankyrase can be regulated has remained unknown. In this study, we propose that phosphorylation at position eight of the TBM enhances the affinity of effectors for the ARC domains of tankyrase. Using isolated TBM peptides, we demonstrate that phosphorylation of serine, but not tyrosine, strengthens ARC binding by up to an order of magnitude. Interrogation of proteome-wide phosphorylation data reveals that phosphorylation at position eight in the TBM is enriched in proteins that support centrosome function/localization. Our findings suggest that TBM phosphorylation may serve as an effector-specific mechanism for tankyrase recruitment/retention, providing an additional layer of regulation to control tankyrase.