Coordinate regulation of the oxygen-dependent degradation domains of hypoxia-inducible factor 1 alpha

Abstract

Oxygen-dependent proteolysis is the primary means of regulating the hypoxia-inducible factor (HIF) family of transcription factors. The alpha-subunit of HIF factor 1 (HIF-1) contains two highly conserved oxygen-dependent degradation domains (402 ODD and 564 ODD), each of which includes a proline that is hydroxylated in the presence of oxygen, allowing the von Hippel-Lindau (VHL) E3 ubiquitin ligase to interact and target HIF-1alpha to the proteasome for degradation. Mutation of either proline is sufficient to partially stabilize HIF-1alpha under conditions of normoxia, but the specific contributions of each hydroxylation event to the regulation of HIF-1alpha are unknown. Here we show that the two ODDs of HIF-1alpha have independent yet interactive roles in the regulation of HIF-1alpha protein turnover, with the relative involvement of each ODD depending on the levels of oxygen. Using hydroxylation-specific antibodies, we found that under conditions of normoxia proline 564 is hydroxylated prior to proline 402, and mutation of proline 564 results in a significant reduction in the hydroxylation of proline 402. Mutation of proline 402, however, has little effect on the hydroxylation of proline 564. To determine whether the more rapid hydroxylation of the proline 564 under conditions of normoxia is due to a preference for the particular sequence surrounding proline 564 or for that site within the protein, we exchanged the degradation domains within the full-length HIF-1alpha protein. In these domain-swapping experiments, prolyl hydroxylase domain 1 (PHD1) and PHD2 preferentially hydroxylated the proline located in the site of the original 564 ODD, while PHD3 preferred the proline 564 sequence, regardless of its location. At limiting oxygen tensions, we found that proline 402 exhibits an oxygen-dependent decrease in hydroxylation at higher oxygen tensions relative to proline 564 hydroxylation. These results indicate that hydroxylation of proline 402 is highly responsive to physiologic changes in oxygen and, therefore, plays a more important role in HIF-1alpha regulation under conditions of hypoxia than under conditions of normoxia. Together, these findings demonstrate that each hydroxylated proline of HIF-1alpha has a distinct activity in controlling HIF-1alpha stability in response to different levels of oxygenation.

FIG. 1.

Proline 564 is hydroxylated prior to proline 402. (A) Protein sequence alignment of HIF-1α, HIF-2α, and HIF-3α subunits for 402 ODD and 564 ODD domains across species demonstrates high conservation. (B) Hydroxylation-specific antibodies recognize only hydroxylated prolines 402 and 564 of HIF-1α. Polyvinyl difluoride membranes were dotted with 10 ng, 100 ng, and 1 μg of hydroxylated (top) and unhydroxylated (bottom) proline 402 or proline 564 peptides and immunoblotted. (C) Nitrocellulose membranes were dotted with twofold serial dilutions of either hydroxylated proline 402 peptide (top) or hydroxylated proline 564 peptide (bottom) and immunoblotted. (D) Constructs transfected into HIF knockout (HKO) mouse embryo fibroblasts (MEFs). The asterisk indicates prolines that are mutated into residues that cannot be hydroxylated. (E) HKO cells were transfected with full-length HIF-1α, P402A HIF-1α, P564G HIF-1α, or empty vector and treated with MG132 (10 μM) for the indicated time or DFO (100 μM, 4 h). Immunoblot analyses were carried out to examine the levels of total HIF-1α, hydroxylated P402 HIF-1α, hydroxylated P564 HIF-1α, and α-tubulin.

FIG. 2.

Proline 564 hydroxylation influences the hydroxylation of proline 402. (A) Constructs transfected into HIF knockout (HKO) mouse embryo fibroblasts (MEFs). The asterisk indicates prolines that are mutated into residues that cannot be hydroxylated. (B and C) Hydroxylation of proline 402 is influenced by hydroxylation of proline 564. HKO MEFs were transfected with full-length HIF-1α or mutant HIF-1α along with the indicated prolyl hydroxylases and treated with MG132 (10 μM). Immunoblots were carried out to determine the levels of total HIF-1α, hydroxylated P402, hydroxylated P564, and α-tubulin. Intensity of exposure was adjusted to illustrate that a small fraction of proline 402 is still hydroxylated in the proline 564 mutant. (D) The effect of endogenous hydroxylases in HKO MEFs was examined. HKO MEFs were transfected with full-length HIF-1α or mutant HIF-1α with and without MG132. Immunoblot analyses were carried out to determine the levels of total HIF-1α, hydroxylated P402, hydroxylated P564, and α-tubulin.

FIG. 3.

Difference in hydroxylation is due to structural determinants in full-length HIF-1α for PHD1 and PHD2, but PHD3 is sequence specific. (A) Oxygen-dependent degradation domain-swapped constructs transfected into HKO MEFs. The asterisk indicates prolines that are mutated to residues that cannot be hydroxylated. (B, C, and D) HKO cells were transfected with degradation domain-swapped mutant HIF-1α and the indicated prolyl hydroxylase. The day following transfection, the cells were treated with MG132 (10 μM) for the indicated time or DFO (100 μM, 4 h). Immunoblot analyses were carried out to examine the levels of total HIF-1α, hydroxylated P402 HIF-1α, hydroxylated P564 HIF-1α, and α-tubulin.

FIG. 4.

Kinetics of hydroxylation of exon-swapped mutant HIF-1α. (A, B, and C) HKO cells were transfected with exon-swapped mutant HIF-1α and the indicated prolyl hydroxylase. The day following transfection, the cells were treated with MG132 (10 μM) for the indicated time or DFO (100 μM, 4 h). Immunoblot analyses were carried out to examine the levels of total HIF-1α, hydroxylated P402 HIF-1α, hydroxylated P564 HIF-1α, and α-tubulin.

FIG. 5.

Single proline point mutants are still targeted for degradation by VHL. (A) HKO cells were transiently transfected with wild-type HIF-1α or mutant HIF-1α. In addition, cells were cotransfected with empty vector or VHL, elongin B, and elongin C. Immunoblot analyses were performed to examine the protein levels of total HIF-1α, VHL, and α-tubulin. (B) HKO cells were transiently transfected with CD fused with wild-type HIF-1α ODD (amino acids 338 to 603) or mutant HIF-1α along with human VHL in increasing amounts of plasmid. Immunoblot analyses were performed to examine the protein levels of cytosine deaminase, VHL, and α-tubulin.

FIG. 6.

HIF-1α is hydroxylated under conditions of mild hypoxia but is still stable. (A) Total cell extracts were prepared from RCC4 without VHL. Cells were treated with the proteasome inhibitor MG132 (10 μM; 1 h), anoxia (>0.2%; 6 h), CoCl₂ (100 μM; 6 h), and/or DFO (100 μM; 6 h). Immunoblot analyses were carried out to show the levels of total HIF-1α, hydroxylated proline 402 of HIF-1α, hydroxylated proline 564 of HIF-1α, and α-tubulin. (B) Total cell extracts were prepared from RCC4 with functional VHL. Cells were treated with the proteasome inhibitor MG132 (10 μM; 1 h), anoxia (>0.2%; 6 h), CoCl₂ (100 μM; 6 h), and/or DFO (100 μM; 6 h). Immunoblot analyses were carried out to show the levels of total HIF-1α, hydroxylated proline 402 of HIF-1α, hydroxylated proline 564 of HIF-1α, and α-tubulin. (C) RCC4 with functional VHL were treated with mild hypoxia (2% or 0.5% O₂) or anoxia for 6 to 24 h and harvested in a hypoxic environment. Cells were also treated with proteasome inhibition for the last hour of hypoxic/anoxic treatment. Total cell extracts were subjected to immunoblot analysis with antibodies to total HIF-1α, hydroxylated proline 402 of HIF-1α, hydroxylated proline 564 of HIF-1α, and α-tubulin.

FIG. 7.

Model of HIF-1α hydroxylation. (A) Under normoxic conditions, proline 564 of HIF-1α is recognized by a prolyl hydroxylase enzyme, leading to hydroxylation of proline 564. HIF-1α can now be recognized by VHL or by another hydroxylase. If VHL interacts, HIF-1α is degraded. Alternatively, hydroxylation of proline 564 enhances the hydroxylation of proline 402. Thus, a PHD can interact with HIF-1α protein hydroxylated at proline 564 to hydroxylate proline 402, providing VHL with two recognition sites to target HIF-1α protein for degradation. (B) As oxygen levels decrease, proline 402 hydroxylation is inhibited prior to proline 564 hydroxylation.