Dysfunction of PINK1, a mitochondrial Ser/Thr kinase, causes familial Parkinson's disease (PD). Recent studies have revealed that PINK1 is rapidly degraded in healthy mitochondria but accumulates on the membrane potential (ΔΨm)-deficient mitochondria, where it recruits another familial PD gene product, Parkin, to ubiquitylate the damaged mitochondria. Despite extensive study, the mechanism underlying the homeostatic control of PINK1 remains unknown. Here we report that PINK1 is autophosphorylated following a decrease in ΔΨm and that most disease-relevant mutations hinder this event. Mass spectrometric and mutational analyses demonstrate that PINK1 autophosphorylation occurs at Ser228 and Ser402, residues that are structurally clustered together. Importantly, Ala mutation of these sites abolishes autophosphorylation of PINK1 and inhibits Parkin recruitment onto depolarized mitochondria, whereas Asp (phosphorylation-mimic) mutation promotes mitochondrial localization of Parkin even though autophosphorylation was still compromised. We propose that autophosphorylation of Ser228 and Ser402 in PINK1 is essential for efficient mitochondrial localization of Parkin.

Background: Parkin is a ubiquitin ligase activated by a decrease in the mitochondrial membrane potential (⌬⌿m). However, details regarding its mechanism remain limited. Results: PINK1-dependent phosphorylation of Parkin at Ser-65 following dissipation of ⌬⌿m triggers ubiquitin-ester transfer by the RING2 domain of Parkin to Cys-431. Conclusion: Parkin catalyzes trans-(ubiquitin-thioester)ification upon PINK1-dependent phosphorylation. Significance: The molecular process of Parkin-catalyzed ubiquitylation has been determined.