Figure 1
From: Mdm2 enhances ligase activity of parkin and facilitates mitophagy
Mdm2 directly binds parkin and enhances its catalytic activity. (A) The left panel shows the load of MBP-parkin (MBP-PK) and MBP control by Coomassie staining. The middle panel shows Western blot for MBP-parkin retained by GST-Mdm2, but not by GST control, detected with anti-MBP antibody. The right panel shows equal loading of bait, GST and GST-Mdm2 (Coomassie staining). (B) Purified MBP-parkin (0.2 μM) was incubated with ubiquitin, without (negative control) or with the mix of E1 + E2 (UbcH7) ligases and indicated concentrations of GST-Mdm2 (0.02–0.05 μM) in 20 μl for 2 h at 30 °C. The reactions were stopped by 20 μl of SDS buffer. The proteins were resolved by SDS-PAGE and blotted with indicated antibodies. MBP blot shows equal loading of MBP-parkin, ubiquitin blot shows stimulation of parkin activity by GST-Mdm2. As reported previously58, only mono-ubiquitination of parkin is detectable in vitro at 30 °C, whereas in cells grown at 37 °C multi- and/or poly-ubiquitination yielding a “ladder” is prevalent (Fig. 2). (C) Quantification of the data shown in (B) from 2 independent experiments. The data were analyzed by one-way ANOVA with Mdm2 as the main factor. *p < 0.001 to NO (buffer in the sample instead of Mdm2) and GST (GST alone 0.05 μM); ap < 0.001 to both 0.05 and 0.035 μM Mdm2; #p < 0.01 to 0.02 μM Mdm2 by Bonferroni host hoc test with correction for multiple comparisons. (D) The domains of parkin and constructs with domain deletions used in the immunoprecipitation experiments. (E) Immunoprecipitation of isolated parkin domains by full-length Mdm2. Left middle panel shows the expression of HA-Mdm2 and Flag-parkin (lane 1 – negative control without parkin) in HEK293 cell lysates. Right middle panel: HA-Mdm2 was IPed with anti-HA antibody and co-IPed Flag-parkin constructs were detected by Western blot with anti-Flag antibody. All constructs containing R2 (full-length WT, arrow; parkin lacking Ubl, double arrow; R1-IBR-R2, white arrow; IBR-R2, white arrowhead) bound Mdm2, whereas R1-IBR did not (detected in lysate, but not in IP sample). Lower panels – no bate (no HA-Mdm2) negative controls. Note that the two bands visible in the negative control are non-specific IgG.
