Stress-induced phospho-ubiquitin formation causes parkin degradation

Lyudmila Kovalchuke; Eugene V. Mosharov; Oren A. Levy; Lloyd A. Greene

L-DOPA induced a significant loss of wild-type overexpressed parkin after 24 hours of treatment, as expected (38.0 ± 2.9% loss, p <0.0001, N = 8) (Fig. 4A,B) . However, the mutants deficient in binding phospho-Ub were significantly resistant to loss compared to wild-type parkin (H302A: 22.9 ± 2.1% loss, N = 8, p = 0.006 vs. WT + L-DOPA; K151E: 20.7 ± 4.3% loss, N = 7, p = 0.003 vs. WT + L-DOPA) (Fig. 4A,B ). Of note, the degree of protection from L-DOPAinduced loss afforded by the H302A and K151E mutations was ~50%, similar to the degree of protection afforded by PINK1 knockdown (Fig 3B) . This suggests that the contribution of PINK1 to L-DOPA-mediated parkin loss may be fully accounted for by parkin's interaction with PINK1-generated phospho-Ub. In agreement with this, the S65A parkin mutant was not at all protected from L-DOPA-induced loss compared to wild-type parkin (S65A: 36.8 ± 3.6% loss, N = 4, p = 0.82) (Fig. 4A,B) , indicating that PINK1-mediated parkin phosphorylation is not . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/484857 doi: bioRxiv preprint required for parkin loss. We also tested whether a protective effect of S65A might be revealed in the context of a parkin mutant that is deficient in binding to phospho-Ub. However, we did not observe greater protection from L-DOPA with a H302A/S65A double mutant over the H302A single mutant (H302A/S65A: 16.1 ± 7.8% loss, N = 3, p = 0.48 vs. H302A alone) (Fig. 4A,B) .