Functional characterization of the Arg371His PHD2 mutant. (A) Association of HIF-2α with Arg371His PHD2. 35S-labeled, in vitro–translated wild-type or Arg371His FlagPHD2 was incubated with 1 μg of either GST or GST–HIF-2α (516-549) immobilized on GSH-agarose. The resins were washed and eluted, and the eluates were subjected to SDS-PAGE and autoradiography. Input represents 10% of the total. The relative recovery of wild-type PHD2 from 3 replicates is 100 ± 24 AU (arbitrary units ± SD), whereas that of Arg371His PHD2 is 2.4 ± 4 AU (P < .005). (B) Association of HIF-1α with Arg371His PHD2. Binding assays with GST–HIF-1α (531-575) were performed as described in panel A. Input represents 5% of total. The relative recovery of wild-type PHD2 from 3 replicates is 100 ± 0.1 AU, whereas that of Arg371His PHD2 is 0.8 ± 0.0001 AU (P < .005). (C) Hydroxylase activity of Arg371His PHD2. Equal quantities (as determined by phosphorimager analysis) of in vitro–translated wild-type or Arg371His FlagPHD2, or mock in vitro translation reaction, was incubated with 0.75 μg of GST–HIF-2α (516-549) for 1 hour in the presence of 2-oxoglutarate, ascorbic acid, and FeCl2. The GST–HIF-2α (516-549) was isolated using GSH-agarose, washed, and then the degree of HIF hydroxylation was assessed by subsequent incubation with 35S-labeled, in vitro–translated VHL. Input represents 5% of the total. Under the conditions of the assay, the recovery of 35S-labeled, in vitro–translated VHL using wild-type PHD2 from 3 independent experiments is 100 ± 38 AU, while that using Arg371His PHD2 is 12 ± 7.6 AU (P < .05). (D) HIF-inhibitory activity of Arg371His PHD2. HEK293 cells were cotransfected with 150 ng of (eHRE)3-Luc, 150 ng of pRL-TK, 300 ng of either pcDNA3 or pSV-Sport-HA-hHIF-2α, and either 0, 0.3, or 0.6 ng of pcDNA3-FlagPHD2 (wild-type or P317R). The total DNA dose was normalized with pcDNA3. At 48 hours after transfection, the cells were harvested and assayed for luciferase activity. Activities were normalized to that of the Renilla luciferase internal transfection control. Shown are results performed in triplicate ± SD. *P < .05; **P < .01 when comparing results of wild-type and mutant PHD2 at the same dose. In separate experiments, HEK293 cells were transfected with 2 μg of wild-type or Arg371His pcDNA3-FlagPHD2; 48 hours later, extracts (15 μg) were analyzed by Western blotting with anti-Flag (M2; Sigma, St Louis, MO) or anti–β tubulin (H-235; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. The position of PHD2, β-tubulin, and a molecular weight marker are as indicated. (E) 3D structure of PHD2. The structure was generated using Cn3D from Protein Data Bank coordinates (2G1M) deposited by McDonough et al.23 Arg371 and Pro317 are highlighted in yellow. Compound A (a 2-oxoglutarate competitive inhibitor) is shown in brown.
