PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, the mechanism by which PINK1 activates and recruits Parkin to mitochondria remains unclear. While PINK1 phosphorylates Parkin, other PINK1 substrates appear to be involved in activating Parkin. Using mass spectrometry, the study found that PINK1 phosphorylates ubiquitin at serine 65, a site homologous to the one phosphorylated on Parkin's ubiquitin-like domain. This phosphorylation of ubiquitin activates Parkin E3 ubiquitin ligase activity. The phosphomimetic ubiquitin mutant S65D bound and activated Parkin, while the mutant S65A inhibited Parkin translocation to damaged mitochondria. These results suggest a feed-forward mechanism where PINK1-mediated phosphorylation of ubiquitin activates Parkin. The study also shows that PINK1 phosphorylates ubiquitin at S65, which is homologous to the site phosphorylated on Parkin's ubiquitin-like domain. This phosphorylation of ubiquitin is essential for Parkin activation and mitochondrial quality control. The findings highlight the role of PINK1 in activating Parkin through the phosphorylation of ubiquitin, which is a key step in the process of mitophagy.PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, the mechanism by which PINK1 activates and recruits Parkin to mitochondria remains unclear. While PINK1 phosphorylates Parkin, other PINK1 substrates appear to be involved in activating Parkin. Using mass spectrometry, the study found that PINK1 phosphorylates ubiquitin at serine 65, a site homologous to the one phosphorylated on Parkin's ubiquitin-like domain. This phosphorylation of ubiquitin activates Parkin E3 ubiquitin ligase activity. The phosphomimetic ubiquitin mutant S65D bound and activated Parkin, while the mutant S65A inhibited Parkin translocation to damaged mitochondria. These results suggest a feed-forward mechanism where PINK1-mediated phosphorylation of ubiquitin activates Parkin. The study also shows that PINK1 phosphorylates ubiquitin at S65, which is homologous to the site phosphorylated on Parkin's ubiquitin-like domain. This phosphorylation of ubiquitin is essential for Parkin activation and mitochondrial quality control. The findings highlight the role of PINK1 in activating Parkin through the phosphorylation of ubiquitin, which is a key step in the process of mitophagy.