Fig. 5
From: Parkin targets HIF-1α for ubiquitination and degradation to inhibit breast tumor progression
Ubquitination of HIF-1α by Parkin at lysine 477. a Left panel: sequences and counts of peptides containing potential ubiquitination sites identified by LC-MS/MS analysis in MCF7 cells with ectopic expression of HA-HIF-1α, Myc-Parkin and His-Ub. *: represents potential lysine ubiquitination site. Right panel: Positions of the top three potential ubiquitination sites in HIF-1α, including K477, K538 and K547. b K477 mutation (K477R) largely abolished ubiquitination of HIF-1α by Parkin. MCF7 cells were transfected with indicated vectors for in vivo ubiquitination assays. c VHL efficiently ubiquitinated K477R but not K532R/K538R/K547R mutant HA-HIF-1α in MCF7 cells analyzed by in vivo ubiquitination assays. d Parkin efficiently ubiquitinated K532R/K538R/K547R but not K477R mutant HA-HIF-1α in MCF7 cells analyzed by in vivo ubiquitination assays. e K477R mutation largely abolished the negative regulation of HA-HIF-1α protein by Myc-Parkin in MCF7 and MDA-MB231 cells. Cells were transduced with vectors expressing WT or K477R HA-HIF-1α together with Myc-Parkin vectors. f Myc-Parkin expression did not affect the K477R HA-HIF-1α protein half-life. MCF7 cells with ectopic Myc-Parkin expression and control cells were transfected with K477R HA-HIF-1α. The data present mean ± S.D. (n = 3). g K477R mutation largely abolished the inhibitory effect of Parkin on HIF-1α luciferase reporter activities in cells. MCF7 and MDA-MB231 cells were transduced with HIF-1α-shRNA #1 to knock down endogenous HIF-1α, and then transduced with vectors expressing WT or K477R HA-HIF-1α resistant to HIF-1α-shRNA (HA-rHIF-1α) (left panels). Cells were then transfected with the HIF-1α luciferase reporter vector together with Myc-Parkin vectors for luciferase reporter assays (right panel). The data present mean ± S.D. (n = 6). **: P < 0.001; two-tailed Student's t test
