The peptidyl prolyl cis/trans isomerase FKBP38 determines hypoxia-inducible transcription factor prolyl-4-hydroxylase PHD2 protein stability

Abstract

The heterodimeric hypoxia-inducible transcription factors (HIFs) are central regulators of the response to low oxygenation. HIF-alpha subunits are constitutively expressed but rapidly degraded under normoxic conditions. Oxygen-dependent hydroxylation of two conserved prolyl residues by prolyl-4-hydroxylase domain-containing enzymes (PHDs) targets HIF-alpha for proteasomal destruction. We identified the peptidyl prolyl cis/trans isomerase FK506-binding protein 38 (FKBP38) as a novel interactor of PHD2. Yeast two-hybrid, glutathione S-transferase pull-down, coimmunoprecipitation, colocalization, and mammalian two-hybrid studies confirmed specific FKBP38 interaction with PHD2, but not with PHD1 or PHD3. PHD2 and FKBP38 associated with their N-terminal regions, which contain no known interaction motifs. Neither FKBP38 mRNA nor protein levels were regulated under hypoxic conditions or after PHD inhibition, suggesting that FKBP38 is not a HIF/PHD target. Stable RNA interference-mediated depletion of FKBP38 resulted in increased PHD hydroxylation activity and decreased HIF protein levels and transcriptional activity. Reconstitution of FKBP38 expression abolished these effects, which were independent of the peptidyl prolyl cis/trans isomerase activity. Downregulation of FKBP38 did not affect PHD2 mRNA levels but prolonged PHD2 protein stability, suggesting that FKBP38 is involved in PHD2 protein regulation.

FIG. 1.

FKBP38 interacts specifically with PHD2. (A) IVTT ³⁵S-labeled PHD1, PHD2, and PHD3 were incubated with GST-HIF-1α-530-826, GST-HIF-2α-404-569, GST-FKBP38, or GST alone. Bound proteins were pulled down and visualized by phosphorimaging. (B and C) Total cell extracts from HEK293 cells transiently transfected with V5-FKBP38 and c-myc-PHD2 were incubated with anti-V5 (B) or anti-c-myc (C) antibodies or with isotype-matched control IgG. The antibodies and bound proteins were immunoprecipitated and analyzed by immunoblotting (WB). Coimmunoprecipitated PHD2 and FKBP38 were detected by specific antibodies. IP, immunoprecipitate. (D) Indirect immunofluorescence of FKBP38 and PHD2. HeLa cells were transfected with a plasmid encoding HA-PHD2 and stained with antibodies to FKBP38 (green) and HA (red), followed by fluorescently labeled secondary antibodies.

FIG. 2.

Mapping of the FKBP38 and PHD2 interaction sites. (A) Schematic representation of the predicted FKBP38 domain architecture and the FKBP38 constructs used. TPR, tetratricopeptide repeats; CaM, calmodulin-binding site; TM, transmembrane domain. (B) IVTT ³⁵S-labeled PHD2 was allowed to interact with GST-FKBP38, GST-FKBP38Δ98-257, GST-FKBP38-3-97, GST-FKBP38-99-412, GST-FKBP38-256-412, or GST alone; the protein complexes were precipitated by glutathione-Sepharose, separated by SDS-PAGE, and visualized by phosphorimaging. (C) IVTT ³⁵S-labeled PHD2-170-426 was tested for interaction with GST-FKBP38, GST-HIF-2α-404-569, or GST alone and analyzed as described above.

FIG. 3.

FKBP38-PHD2 interaction and FKBP38 gene expression are not regulated by oxygen. (A) HeLa cells were transiently transfected with Gal4-DBD and VP16AD fusion protein vectors and Gal4 response element-driven firefly luciferase reporter, as well as a Renilla luciferase control vector. Following transfection, the cells were incubated under normoxic (20% O₂) or hypoxic (0.2% O₂) conditions, and luciferase reporter gene activities were determined 16 h later. Firefly/Renilla luciferase activity ratios were normalized to the normoxic negative control DBD-p53/AD-CP cotransfection, which was arbitrarily defined as 1. Mean values plus standard errors of the mean are shown for three independent experiments performed in triplicate. (B and C) HeLa cells were cultured under normoxic (20% O₂) or hypoxic (0.2% O₂) conditions or treated with the PHD inhibitor dimethyloxalylglycine (DMOG) (2 mM). (B) Total RNA was extracted after 8 h of incubation, and mRNA levels of FKBP38, PHD1 to -3, GLUT1, CAIX, and ribosomal protein L28 were quantified by real-time RT-PCR. The transcript levels of these genes were normalized to L28, and hypoxic inductions were calculated (mean, n = 2). (C) Cellular proteins were extracted and separated by SDS-PAGE, and endogenous FKBP38, PHD2, HIF-1α, and β-actin levels were analyzed by immunoblotting.

FIG. 4.

Enhanced hydroxylation activity by RNAi-mediated FKBP38 depletion. HeLa cells were transfected with vectors containing FKBP38 RNAi or control oligonucleotides, and stable clones were selected (3A5, 3D6, and 2G8). (A) Total RNA was extracted, and FKBP38 transcript levels were determined by real-time RT-PCR and normalized to L28 mRNA levels (mean, n = 2). (B) Total cell extracts were prepared and analyzed by immunoblotting for FKBP38 and β-actin expression. Total cell extracts of untransfected (C) and FKBP38-reconstituted (D) stable RNAi clones were prepared, and hydroxylation activity was measured using the VBC-binding assay. Shown are mean values of relative VBC binding plus standard errors of the mean of three independent experiments performed in triplicate. P values were obtained by paired t tests (*, P < 0.05). Subsequently, cell extracts were analyzed by immunoblotting for endogenous FKBP38, transfected V5-FKBP38, and β-actin expression. wt, wild type; ctrl, control.

FIG. 5.

FKBP38 regulates HIF-dependent reporter gene activity. (A) HeLa wild-type (wt) as well as FKBP38 siRNA cell clones were cotransfected with pH3SVL and pRL-SV40 Renilla luciferase reporter vectors and cultivated for 16 h under normoxic (20% O₂) or hypoxic (0.2% O₂) conditions before relative luciferase activities were determined. The results are mean values plus standard errors of the mean of five independent experiments performed in triplicate. ctrl, control. (B) Stable FKBP38 RNAi HeLa clones were transiently transfected with Gal4-DBD-HIF-1α-370-429-VP16AD expression vectors (schematically represented) and Gal4 response element-driven firefly luciferase reporter, as well as a Renilla luciferase control vector. (C) HeLa clones were cotransfected with the one-hybrid reporter gene vectors and FKBP38, FKBP38-99-412, or lacZ control expression vectors. Eight hours posttransfection, the cells were cultured under either normoxic or hypoxic conditions for an additional 16 h, and firefly luciferase activities were determined and corrected for Renilla luciferase activity. The results are mean values of relative luciferase activities plus standard errors of the mean of at least three independent experiments performed in triplicate. P values were obtained by paired t tests (**, P < 0.01; *, P < 0.05). Expression of the transfected V5-tagged vectors was verified by immunoblotting against V5 and β-actin.

FIG. 6.

Increased PHD2 protein stability in FKBP38-depleted cells. (A) Wild-type HeLa cells (wt), stable FKBP38-silenced HeLa clones, V5-FKBP38 reconstituted HeLa cells, or FKBP38-silenced HeLa cells treated with the FKBP38 inhibitor GPI1046 (10 μM) were cultivated under normoxic (20% O₂) conditions, and FKBP38, V5-FKBP38, PHD2, or β-actin was detected by immunoblotting. (B) Total RNA was extracted and reverse transcribed into cDNA, and mRNA levels of PHD1, PHD2, and PHD3 were normalized to ribosomal L28 mRNA levels as quantified by real-time RT-PCR (n = 2). ctrl, control. (C) Total hypoxic FKBP38 RNAi cell clone lysates were separated by SDS-PAGE and analyzed for FKBP38, PHD2, HIF-1α, and β-actin protein levels by immunoblotting. (D) Total RNA was extracted after 8 h of incubation in normoxia or hypoxia, and mRNA levels of FKBP38, PHD1 to -3, GLUT1, and ribosomal L28 were quantified by real-time RT-PCR. The transcript levels of these genes were normalized to L28 mRNA, and hypoxic inductions were calculated (n = 2).

FIG. 7.

Increased PHD2 protein stability in FKBP38-silenced cells. (A) The half-lives of PHD2 protein in wild-type HeLa (wt) and FKBP38-depleted (2G8) cells were monitored by a pulse-chase experiment. Cells were metabolically labeled with [³⁵S]Met/Cys for 2 h, followed by chase for the indicated times. PHD2 was immunoprecipitated from the cell lysates with anti-PHD2 antibodies, and signals were detected by autoradiography. Translation was blocked in FKBP38 RNAi HeLa clones by addition of 100 μM cycloheximide and PHD2 (B), as well as FKBP38 (C), and protein levels were determined after the indicated time periods by immunoblotting. Subsequent detection of β-actin served as a loading control. (D) Total lysates of HeLa cells were prepared at the indicated periods of hypoxia or hypoxia followed by reoxygenation, and PHD2 as well as β-actin levels were detected by immunoblotting.