Binding to serine 65‐phosphorylated ubiquitin primes Parkin for optimal PINK1‐dependent phosphorylation and activation

Abstract

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser 65 )—which lies within its ubiquitin‐like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser 65 . How Ser 65 ‐phosphorylated ubiquitin (ubiquitin Phospho‐Ser65 ) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin Phospho‐Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser 65 by PINK1 in vitro . Analysis of the Parkin structure, corroborated by site‐directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin Phospho‐Ser65 , thereby promoting Parkin Ser 65 phosphorylation and activation of its E3 ligase activity in vitro . Mutation of His302 markedly inhibits Parkin Ser 65 phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin Phospho‐Ser65 to Parkin disrupts the interaction between the Ubl domain and C‐terminal region, thereby increasing the accessibility of Parkin Ser 65 . Finally, purified Parkin maximally phosphorylated at Ser 65 in vitro cannot be further activated by the addition of ubiquitin Phospho‐Ser65 . Our results thus suggest that a major role of ubiquitin Phospho‐Ser65 is to promote PINK1‐mediated phosphorylation of Parkin at Ser 65 , leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho‐Ser 65 ‐binding pocket on the surface of Parkin that is critical for the ubiquitin Phospho‐Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin Phospho‐Ser65 , which could aid in the development of Parkin activators that mimic the effect of ubiquitin Phospho‐Ser65 .