Stress-induced phospho-ubiquitin formation causes parkin degradation

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

Association with L-DOPA-induced phospho-Ub leads to parkin loss Having implicated PINK1 in L-DOPA-induced parkin depletion, we next sought to uncover the mechanism by which PINK1 contributes to this loss. PINK1 activates parkin by phosphorylating Ser65 on both parkin and on ubiquitin [42] [43] [44] , [49] , [72] [73] [74] [75] [76] [77] , with parkin binding the latter non-covalently. Parkin binds to both unconjugated phospho-mono-Ub and to . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 phospho-poly-Ub chains [43] , [78] , [79] . We reasoned that parkin phosphorylation, parkin binding to L-DOPA-induced phospho-Ub, or both of these events may underlie PINK1-mediated parkin loss. To determine the relative importance of these events, we introduced several point mutations in the "moderately" overexpressed exogenous parkin construct described above (Fig. S4A ). To test the importance of parkin phosphorylation, we generated a S65A parkin mutant. To test the importance of phospho-Ub binding to parkin, we generated parkin mutants H302A and K151E. Both of the latter mutations significantly abrogate the interaction between parkin and phospho-Ub, although K151E has been demonstrated to be more effective in this regard [47] . We delivered the parkin mutants to PC12 cells via lentiviral transduction and tested their responses to L-DOPA treatment. Although the viral titers between constructs varied slightly due to inherent variability in the viral production process, we found that all parkin mutants used in this study were expressed at similar levels (i.e. ~8-fold higher than endogenous parkin) in cells when controlling for titer (Fig. S7 ).