Figure 9.

PTENα promotes PRKN self-association. (a) PRKN interacts with itself through its C-terminus. Plasmids encoding PRKN-FLAG and its truncations were co-transfected with a plasmid encoding PRKN-GFP into HEK293T cells. Cells were treated with 10 μM CCCP for 1 h before harvest. Lysates immunoprecipitated with FLAG M2 beads were immunoblotted with anti-GFP or anti-FLAG. (b) PTENα promotes self-association of PRKN. Plasmids encoding PRKN-GFP, PRKN-FLAG and PTENα-GFP were co-transfected into HEK293T cells as indicated. Cells were treated with 10 μM CCCP for 1 h before harvest. Cell homogenates were immunoprecipitated with anti-FLAG M2-conjugated beads and immunoblotted with anti-FLAG and anti-GFP. (c) Co-IP in PTENα^+/+or PTENα^-/- HeLa cells co-transfected with plasmids encoding PRKN-GFP and PRKN-FLAG. Cells were treated with 20 μM CCCP or DMSO for 2 h before harvest. Lysates immunoprecipitated with anti-FLAG M2-conjugated beads were immunoblotted with anti-GFP or anti-FLAG. (d) Self-association of PRKN in HEK293T cells co-transfected with plasmids encoding PTENα and its truncations. Cells were treated with 10 μM CCCP for 1 h before harvest. Cell homogenates were immunoprecipitated with anti-FLAG M2-conjugated beads and immunoblotted with anti-FLAG and anti-GFP. GAPDH was used as a loading control. PRKN self-association was quantified with ImageJ (n = 3). **p < 0.01; ***p < 0.001compared with the group that was transfected with empty vector, analyzed with the two-tailed paired Student’s t-test. (e) A graphical model for PTENα regulation of mitophagy through PRKN. Under stress, PTENα recruits PRKN onto depolarized mitochondria through protein interaction. PTENα amplifies the effect of this recruitment by promoting self-association of PRKN, leading to mitophagy.