Stress-induced phospho-ubiquitin formation causes parkin degradation

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

To promote turnover of cellular proteins, PINK1 must be activated, and a role for this kinase in parkin turnover in response to L-DOPA thus suggests that the latter causes PINK1 activation. To assess this, we examined S65-phosphorylated ubiquitin (phospho-Ub) as a readout of PINK1 activity. At baseline, phospho-Ub is present at very low or undetectable levels in cells [69] , [70] . Following mitochondrial depolarization, PINK1 becomes stabilized on the outer mitochondrial membrane and activated, leading it to phosphorylate ubiquitin and resulting in an accumulation of phosphorylated poly-ubiquitin chains (phospho-poly-Ub) that is detectable by Western blotting [69] , [70] . As expected, we observed little phospho-Ub in untreated PC12 cells (Fig. 3C) . As a positive control for PINK1 activation and phospho-Ub induction, we treated cells with the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which is widely used as a mitochondrial depolarizing agent [71] . Following CCCP treatement, we observed the appearance of high molecular weight phospho-Ub ladders, consistent with formation of phosphopoly-Ub chains (Fig. 3C ). Treatment of cells with L-DOPA also led to formation of phosphopoly-Ub (Fig. 3C) , although the strength of the L-DOPA-induced phospho-poly-Ub signal was about 10 times weaker than that induced by CCCP (p = 0.04, N = 3 for L-DOPA and 4 for CCCP) (Fig. 3C,D) . We did not observe the formation of unconjugated mono-phospho-Ub after L-DOPA treatment (Fig. 3C) . Knockdown of PINK1 strongly attenuated the phospho-poly-Ub signal from CCCP exposure (to 27.5 ± 5.1% of sh-ctrl, p = 0.003, N = 4), and caused a similar downward trend in the L-DOPA-induced phospho-poly-Ub signal (to 40.5 ± 16.3% of sh-ctrl, p = 0.07, N = 3), indicating that PINK1 indeed appears to be involved in generating this signal ( Fig. 3E ,F). Together, these results indicate that L-DOPA exposure leads to PINK1 activation, albeit to a lesser extent than CCCP exposure, and that this activation contributes to parkin loss.