Stress-induced phospho-ubiquitin formation causes parkin degradation

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

The above findings indicate that parkin's interaction with phospho-Ub is required for a portion of its loss following L-DOPA treatment, while parkin phosphorylation appears to be dispensable. However, one potential reservation for this interpretation is that our mutant parkin constructs have an N-terminal tag to differentiate them from endogenous parkin, and such tags have been reported to disrupt parkin's autoinhibited conformation by opening the Ubl domain [80] . Because parkin phosphorylation is also reported to open the Ubl domain [45] , [73] , [81] [82] [83] [84] , we wished to ensure that the effect of parkin phosphorylation was not made functionally redundant by the presence of the tag. To do so, we took advantage of the finding that parkin phosphorylation is required for full parkin activation in response to CCCP [49] , [77] . We compared the levels of CCCP-induced phospho-poly-Ub in cells transduced with either wildtype or S65A parkin. We reasoned that since parkin phosphorylation contributes to its activation and since such activation leads to phospho-poly-Ub formation following mitochondrial depolarization [50] , we would expect to see decreased levels of phospho-poly-Ub in CCCP- 4A) suggest that parkin loss from L-DOPA treatment occurs via two distinct pathways, and that oxidative stress and PINK1 activity each play a role in only one of these pathways. To determine whether the oxidative stress-dependent (glutathione-sensitive) and the phospho-Ub-dependent mechanisms of parkin loss are in the same pathway, we investigated whether glutathione can abrogate the phospho-Ub signal induced by L-DOPA. Given that the majority of this signal is present in high molecular weight phospho-poly-Ub conjugates, we examined the effect of glutathione on phospho-poly-Ub. We found that glutathione prevented L-DOPA-induced phospho-poly-Ub formation almost completely (no treatment: 28.6 ± 7.8% phospho-poly-Ub relative to L-DOPA treatment, p = 0.002, N = 5; L-DOPA + GSH: 37.6 ± 5.6% relative to L-DOPA treatment, p = 0.002, N = 5) (Fig. 4C) . Additionally, hydrogen peroxide exposure induced phospho-poly-Ub formation (Fig. 4D) . These results indicate that oxidative stress from L-DOPA exposure leads to PINK1 activation and that this in turn leads to the formation of phospho-Ub, its association with parkin, and parkin loss. 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