Figure 3

Direct interactions between Parkin and TRAF2/6. (a) Interaction between TRAF2 and Parkin. IP analysis was performed to determine the binding between TRAF2 and Parkin. PUMA was used as a negative control. TRAF2 selectively interacted with Parkin. (b) The middle region of Parkin is responsible for its interaction with TRAF6. GST-Parkin deletion fragments were incubated with TRAF6-transfected 293 lysates for 30 min at room temperature. TRAF6 that associated with GST-tagged proteins was detected by WB analysis. Arrowheads indicate the expected sizes of the GST-fused Parkin fragments. (c) The N-terminal RING domain of TRAF6 is responsible for Parkin binding. Two types of recombinant TRAF6 (RING domain; 50–159 amino acids (aa), TRAF domain; 346-504 aa) were used for binding with Parkin, and the RING domain was co-immunoprecipitated with Parkin using a GST-pull down. (d) The N-terminal region of TRAF6 is ubiquitinated by Parkin. Recombinant TRAF6 proteins (N-terminal domain, TRAF6-N; C-terminal TRAF6, TRAF6-C) were incubated with EV- or Parkin-transfected 293 cell lysates (which had already been transfected with HA-Ub) for 6 h at room temperature. Ub-TRAF6 was detected by WB analysis with an HA antibody. (e) The TRAF6 residues R91 and R96 are ubiquitination sites. TRAF6 mutants (K91R, K96R and K104R) were incubated with 293 cell lysates that were co-transfected with HA-Ub and Parkin or EV. Ubiquitinated TRAF6 was detected by WB analysis with an HA antibody. The lysine residues (red) were replaced by arginine (lower panel). (f) TRAF6-K91R is resistant to Parkin-induced TRAF6 suppression. Recombinant TRAF6 proteins (WT and K91R) were delivered by liposomes into 293 cells that had been transfected with EV or Parkin. After 6 h, existing proteins were detected by WB analysis with an anti-GST antibody. When cells were treated with ALLN, GST-TRAF6-WT levels were not reduced in Parkin-transfected cells.