Binding to serine 65‐phosphorylated ubiquitin primes Parkin for optimal PINK 1‐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 (Ser65)—which lies within its ubiquitin-like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 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 ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 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 ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of ubiquitinPhospho-Ser65.

Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation

“…A ubiquitin-derived peptide (12-27 aa) was present only in the crosslinked samples, indicating that these bands contained ubiquitin. These results indicate that the spatial coordinates of ubiquitin Ile 36 are in close proximity to the IBR 350 -366-aa region of Parkin, and that ubiquitin Arg 42 and Arg 72 are in close proximity to 315-334 aa, which corresponds to the RING1-IBR junction of Parkin. Of note, BPA introduced into these peptides, at 355, 356, 357, 327, and 329, was crosslinked to ubiquitin with high efficiency (Figs.…”

“…The Muqit group (42) identified Parkin Lys 151 /His 302 residues as critical residues for binding phosphorylated ubiquitin. Of note, two of the four Parkin residues (Lys 151 , His 302 , Arg 305 , and Gln 316 ) that form a phosphate binding pocket were identified in our computational modeling approach.…”

“…We finally constructed superposition model of Parkin and ubiquitin binding by computational modeling. Based on the crosslinking experiments that were coupled to mass spectrometry, we constructed the first model in which ubiquitin Ile 36 , Arg 42 , and Arg 72 residues are in close proximity to the IBR and RING 1 domains (Fig. 6C).…”

Ubiquitin chain diversity at a glance

“…Phospho-ubiquitin has been linked to mitochondrial quality control through mitophagy and, in this context, to neurological disorders such as Parkinson's disease. The protein kinase PINK1 phosphorylates residue S65 of ubiquitin, which is required for allosteric activation of the ubiquitin ligase Parkin and is essential for the recruitment of the autophagic machinery, including autophagy receptors (Kane et al, 2014;Kazlauskaite et al, 2014Kazlauskaite et al, , 2015Koyano et al, 2014;Kumar et al, 2015;Lazarou et al, 2015;Ordureau et al, 2014;Sauvé et al, 2015;Swaney et al, 2015;Wauer et al, 2015a,b). These events promote the assembly of K6-, K11-, K48-and K63-linked ubiquitin chains on numerous MOM proteins by Parkin (Cunningham et al, 2015;Ordureau et al, 2014;Sarraf et al, 2014).…”

How to get rid of mitochondria: crosstalk and regulation of multiple mitophagy pathways

“…Phosphoubiquitin has a high affinity to Parkin and induces a displacement of the UBL of Parkin, leading to the unveiling of its ligase active domain (Kazlauskaite et al, 2015;Kumar et al, 2015;Sauve et al, 2015;Wauer et al, 2015a;Yamano et al, 2015;Aguirre et al, 2017). The displaced UBL is now more accessible for the Pink1-driven phosphorylation, stabilizing the 'open' state of Parkin.…”