Stress-induced phospho-ubiquitin formation causes parkin degradation

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

To examine this possibility, we compared the levels of stress-induced phospho-poly-Ub in PC12 cells transduced with our "moderately overexpressed" wild-type parkin, a catalytically inactive parkin mutant (C431S), or an empty control vector. In CCCP-treated cells, expression of wild-type parkin increased the phospho-poly-Ub signal by about 3-fold over that of cells expressing empty vector, as anticipated (WT: 2.85 ± 0.30 relative to empty vector, p = 0.01, N = 5) (Fig. 8A,B ). This increased phospho-poly-Ub signal can be attributed to parkin activity because catalytically inactive parkin almost completely abrogated the increased phospho-poly- The lack of evidence for parkin-dependent phospho-poly-Ub formation following L-DOPA and hydrogen peroxide treatment suggested that mitochondrial parkin activity is not necessary for its phospho-poly-Ub-dependent loss. Indeed, L-DOPA exposure decreased catalytically inactive C431S parkin levels to the same extent as it did with wild-type parkin (WT: