Figure 3
From: Mdm2 enhances ligase activity of parkin and facilitates mitophagy
Mdm2-dependent increase of parkin ubiquitination requires catalytically active parkin. (A) HEK293 cells were transfected with myc-parkin or catalytically inactive myc-parkin-T415N mutant and HA-Mdm2. Mdm2 was immunoprecipitated with anti-HA antibody, and the samples were blotted for HA and myc. The results (left panel) show that both WT and mutant parkin interact with Mdm2. Right panel shows the expression of myc-parkin constructs and HA-Mdm2 in cell lysates. (B) HEK293A cells were transfected with HA-ubiquitin, myc-parkin or catalytically inactive myc-parkin-T415N mutant, and varying amounts of untagged Mdm2. Parkin was immunoprecipitated with anti-myc antibody, and its ubiquitination was determined by Western blot with anti-HA antibody. Upper panels show the expression of myc-parkin and untagged Mdm2 in cell lysates. Lower panel shows ubiquitination of parkin detected with anti-HA antibody in immunoprecipitated parkin (upper blot) and the equal load of immunoprecipitated myc-parkin detected with anti-myc antibody. Note that Mdm2 progressively increases the ubiquitination of WT parkin, but has no effect on catalytically inactive T415N mutant, which binds Mdm2 as well as WT parkin, demonstrating that Mdm2 enhances parkin self-ubiquitination.
