PMC

Model for interaction of PHD2 with HSP90 pathway. PHD2 associates with p23 via a PXLE motif in p23 (denoted by curved line). p23 in turn is known to bind to the N terminus of HSP90. This facilitates PHD2-catalyzed hydroxylation of HIF-1α, which is known to be an HSP90 client protein. Prolyl hydroxylation in turn marks HIF-1α for degradation. The C terminus of HSP90 is known to bind a subset of HSP90 co-chaperones that includes FKBP38, FKBP51, and FKBP52. FKBP38 possesses a PXLE motif and, therefore, provides an independent means for recruiting PHD2 to the HSP90 machinery.

The MYND-type zinc finger of PHD2 is required for its interaction with p23. A and B, HEK293FT were transfected and immunoprecipitated (IP) as in B. WB, Western blot. C, shown is a schematic diagram of PHD2. Dark shading denotes the N-terminal MYND-type zinc finger (ZF), light shading denotes the C-terminal prolyl hydroxylase (PH) domain, and numbers indicate amino acid number. Shown at the top are amino acids 21–58. Underlining indicates predicted zinc chelating residues. Inverted triangles indicate Cys-36 and Cys-42.

Knockdown of p23 augments HIF-1α protein levels. A, the indicated cell lines were treated with the indicated siRNA and then maintained under normoxia (NX) or subjected to 2% O₂ (HX) for 4 h. Cell lysates were prepared, and equal protein amounts were then subjected to Western blotting using antibodies to the indicated proteins. Con = control non-targeting siRNA #3. The p23 siRNA was PTGES3_1. B, HeLa cells were treated with the indicated siRNA and then examined as in A. The p23-1 siRNA was PTGES3_1, and the p23-2 siRNA was PTGES3_2.

A conserved PXLE motif in select HSP90 co-chaperones. A, shown is a schematic diagram of p23. Shading denotes the CHORD and Sgt1 (CS) domain, and numbers indicate amino acid number. The C-terminal 20 amino acids of p23 are shown and compared with the indicated residues of FKBP38. B, shown is a comparison p23 sequences from Homo sapiens (Unigene Hs.50425), Mus musculus (Mm.305816), Xenopus. laevis (Xl.14340), Danio rario (Dr.77365), C. elegans (Cel.8134), and Trichoplax adhaerens (XP_002111186). Shading indicates residues conserved in all of the proteins shown. C, HEK293FT were transfected and immunoprecipitated (IP) as in panel B. AAA denotes a P157A/L159A/E160A mutation in p23. WB, Western blot.

PHD2 binds to a PXLE motif. A, shown is a comparison of p23 residues 151–160 to FKBP38 residues 47–56. The numbers above the sequences correspond to p23 residues. Shading indicates conserved residues of the PXLE motif. B, Sf9 lysates containing His₆-FLAG-PHD2 were incubated with p23 peptides immobilized on streptavidin-agarose and washed, and the eluates were examined for the presence of PHD2 by Western blotting using anti-FLAG antibodies. Amino acid substitutions are indicated in each lane. PLE>AAA denotes P157A/L159A/E160A, and DDE>AAA denotes D152A/D153A/E154A. Experiments with FKBP38 peptides are shown in the last two lanes of the bottom right panel. FKBP38 AAA denotes P53A/L55A/E56A. In each panel, peptide was omitted in the first lane. The relative degree of binding for each peptide, normalized to that of wild-type (WT) p23 peptide, is given below each panel.

The PHD2-p23 interaction facilitates prolyl hydroxylation of HIF-1α. A, HeLa cells were treated with control (Con) or p23 siRNA, and cellular extracts were prepared. GST or GST-HIF-1α (531–575) prebound to GSH-agarose was incubated with the extracts and washed, and bound PHD2 was then assessed by Western blotting (WB) using the indicated antibodies. B, PC3 cells were treated with the indicated concentrations of 17-AAG and then immediately subjected to normoxia (NX) or 2% O₂ for 4 h. Cell lysates were prepared, and equal protein amounts were subjected to Western blotting using antibodies to the indicated proteins. C, RCC4 cells were treated with the indicated concentrations of 17-AAG and then immediately subjected to normoxia or 2% O₂ for 4 or 20 h. Cell lysates were prepared, and equal protein amounts were subjected to Western blotting using antibodies to the indicated proteins. D, RCC4 cells were subjected to either 6 nm 17-AAG, 2% O₂ (HX), or both for 20 h. The cells were lysed, and equal protein amounts were incubated with or without recombinant FLAG-VHL, the FLAG-VHL was immunoprecipitated, and proteins were captured by VHL analyzed by Western blotting using antibodies to HIF-1α. Lysates were also analyzed by Western blotting. E, HeLa cells were treated with the indicated siRNA and then subjected to 2% O₂ for 4 h in the presence of 10 μm MG132. The cells were lysed, equal protein amounts were incubated with or without recombinant FLAG-VHL, the FLAG-VHL was immunoprecipitated, and proteins were captured by VHL analyzed by Western blotting using antibodies to HIF-1α. Lysates were also analyzed by Western blotting. F, HeLa cells were treated with the indicated siRNA and then subjected to 2% O₂ for 4 h in the presence of 10 μm MG132. The cells were lysed, equal protein amounts were incubated with recombinant FLAG-VHL, the FLAG-VHL was immunoprecipitated, and proteins were captured by VHL analyzed by Western blotting using antibodies to HIF-1α. Lysates were also analyzed by Western blotting.

Knockdown of p23 augments HIF-1α target genes. A–E, HeLa cells were treated with the indicated siRNA for 72 h and then maintained under normoxia (NX) or subjected to 2% O₂ (HX) for 16 h. mRNA levels for the indicated genes were measured by real time PCR and normalized to that of 18 S rRNA. Shown are the means ± S.D., n = 3. *, p < 0.05 compared with the respective normoxic or hypoxic control; **, p < 0.01 compared with the respective normoxic or hypoxic control; ns, not significant compared with the respective normoxic or hypoxic control. F, HEK293FT cells were transfected with 50 ng of (eHRE)3-Luc, 50 ng of pRL-TK, and either 5 or 15 ng of either pcDNA3-FLAG-PHD2 or pcDNA3-HA-PHD2 (C36S/C42S). DNA doses were held constant by the addition of pcDNA3. After 8 h, cells were exposed to 1% O₂ (HX) for an additional 16 h or maintained under normoxia (NX). Cells were lysed, and luciferase activity was measured and normalized to that of Renilla luciferase. Shown are the means ± S.D., n = 3. *, p < 0.05 compared with the respective normoxic or hypoxic control; **, p < 0.01 compared with the respective normoxic or hypoxic control; ns, not significant compared with the respective normoxic or hypoxic control.

Mass spectrometry identification of PHD2-interacting proteins. A, Flp-In T-Rex HEK293 or Flp-In T-Rex HEK293/FLAG-PHD2 cells were grown in the presence of amino acids with light or heavy labeled lysine/arginine, respectively and subjected to immunoprecipitation (IP) with anti-FLAG antibodies, and the eluates were mixed and subjected to tryptic digest and mass spectrometry. The square and hexagon depict proteins that bind specifically to PHD2, whereas the small circles depict proteins that bind non-specifically to the antibody (or resin). B, HEK293FT cells were transfected with expression vectors for the indicated proteins, lysed, and subjected to immunoprecipitation with anti-FLAG antibodies, and Western blots (WB) were then performed. The positions of HA-PHD2 or molecular weight markers are shown to the right. C, Flp-In T-Rex HEK293 or Flp-In T-Rex HEK293/FLAG-PHD2 cells were induced with doxycycline, lysed, and subjected to immunoprecipitation with anti-FLAG antibodies. The immunoprecipitates were eluted with 3X FLAG peptide. Aliquots of lysate or eluate were then subjected to Western blotting using antibodies against the indicated proteins. Anti-FLAG antibodies were employed to detect FLAG-PHD2. D and E, HEK293FT cells were maintained under normoxia or hypoxia (1% O₂ for 4 h) (D) or treated with control or p23 siRNA (E). Ab, antibody. Cells were lysed, incubated with 10 μg of either control or anti-PHD2 monoclonal antibody, and immunoprecipitated with protein G-agarose, and Western blots were then performed.