FIGURE 5.

E3 ligase activity-dependent autophagy activation by parkin. A, E3 ligase activity deficient K161N mutant fails to regulate autophagy under normal conditions and starvation treatment (HBSS for 1 h). 293 cells expressing FLAG, FLAG-parkin, or FLAG-parkin (K161N) were subjected to immunoblot analysis with antibodies as indicated. B, Parkin, but not K161N mutant, mono-ubiquitinates Bcl-2 and increases its interactions with Beclin 1. 293 cells expressing a combination of EGFP-Bcl-2, HA-ubiquitin, and FLAG or FLAG-parkin (WT or K161N mutant) were subjected to immunoprecipitation using anti-GFP antibody. Immunoprecipitants and inputs were detected with antibodies as indicated. IP, immunoprecipitation.