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Leu-574 of human HIF-1 is a molecular determinant of prolyl hydroxylation ¹

Laboratory of Human Carcinogenesis, NCI, National Institutes of Health, Bethesda, Maryland, USA; and
^* The Henry Wellcome Building of Genomic Medicine, Oxford, UK

² Correspondence: Laboratory of Human Carcinogenesis, NCI, National Institutes of Health, Bethesda, MD 20892, USA. E-mail: huange{at}mail.nih.gov

SPECIFIC AIMS

HIF-1 is constantly degraded in normoxia via the ubiquitin–proteasome pathway. The von Hippel-Lindau (VHL) E3 ubiquitin ligase binds HIF-1 through specific recognition of hydroxyprolines that are modified by the oxygen-dependent HIF prolyl hydroxylases (PHDs/HPHs). Despite the identification of a conserved Leu-X-X-Leu-Ala-Pro motif, the molecular requirement of HIF-1 for PHDs/HPHs binding remains elusive. We recently demonstrated that Leu-574 of human HIF-1, 10 residues downstream of Pro-564, is essential for VHL recognition and protein degradation. In light of its nonessential role in direct contact with the VHL protein, we investigated the involvement of Leu-574 in prolyl hydroxylation and PHD/HPH recruitment.

PRINCIPAL FINDINGS

1. Leu-574 is required for HIF-1 instability
We have reported that mutation or deletion of Leu-574 in human HIF-1 gave rise to the stabilization of carboxyl-terminal, oxygen-dependent degradation domain (C-ODD, amino acids 498-603), resulting from the loss of VHL binding. To confirm the destabilizing nature of Leu-574, we substituted Leu-574 with serine in full-length HIF-1. It has been shown that Pro-402 and Pro-564 are independently recognized by the VHL ubiquitin ligase. Therefore, mutation of Pro-402 only modestly increased HIF-1 levels in normoxia. Similar to a single Pro-564 mutation in full-length HIF-1, mutation of Leu-574 alone hardly affected HIF-1 expression. Use of a greater amount of plasmids gives rise to constitutive expression of wild-type HIF-1, presumably due to saturation of the proteasome system. The above result suggests that our transfection conditions were appropriate even though a single mutation of Pro-402, Pro-564, or Leu-574 did not stabilize HIF-1 in normoxia. However, mutations of Leu-574 and Pro-402 significantly enhanced HIF-1 stability in normoxia, resulting in constitutive expression of the protein and significant reduction of the inducibility by desferrioxamine (DFO), an iron chelator. A similar expression pattern was observed when Pro-402 and Pro-564 were mutated, indicating that Leu-574 participates in the same pathway as Pro-564 for HIF-1 degradation.

The role of Leu-574 in HIF-1 degradation was further examined in an HIF-1-mediated reporter system. Similar to the Pro-402 and Pro-564 double mutant, the Pro-402 and Leu-574 double mutant exhibited a significant increase in transcriptional activity, especially in normoxia. The transcriptional activity of both mutants remained hypoxia inducible, despite similar protein levels of normoxic and hypoxic samples. This discrepancy may be because FIH-1 inhibits the transcriptional activity of HIF-1 in normoxia whereas endogenous HIF-1, in addition to exogenously expressed HIF-1 mutants, enhances the reporter activity only in hypoxia, thereby maintaining transcriptional inducibility. Mutation of Leu-574 alone also elevated reporter activity. These results, together with our previous report, support that Leu-574 of HIF-1 is required for C-ODD proteolysis by facilitating VHL binding, resulting in HIF-1 degradation.

2. Mutation of Leu-574 inhibits prolyl hydroxylation
Recent structural studies of VHL-HIF-1 interaction indicate that Hyp-564 of HIF-1 is required for VHL recognition whereas the neighboring amino acid residues only contribute to the complex’s stability through a ß sheet-like contact. These studies showed that Leu-574 is nonessential when Pro-564 is artificially hydroxylated, suggesting involvement of Leu-574 in the upstream event(s), such as prolyl hydroxylation. To test this, we raised polyclonal rabbit antisera against a synthetic peptide corresponding to amino acids 558-569 of HIF-1 in which Pro-564 is hydroxylated, and termed the resultant antibody anti-Hyp-564.

To test the epitope specificity of the antibody, we transfected full-length HIF-1 and its Pro-402 and Pro-564 mutants into 293 cells (containing low levels of endogenous HIF-1) in the presence of a proteasome inhibitor Cbz-LLL. As shown in Fig. 1 a, the antibody detected HIF-1 wild-type and the Pro-402 mutant, but not those containing mutated Pro-564 (top panel). In contrast, an anti-HIF-1 antibody (recognizing HIF-1 amino acids 610-727) revealed all the HIF-1 variants (bottom panel), indicating the requirement of Pro-564 as part of the epitope. To demonstrate the antibody specificity for recognizing hydroxylated Pro-564, we resorted to an in vitro translation system in which either DFO or FeCl₂ was added to generate nonhydroxylated or hydroxylated HIF-1. The anti-Hyp-564 antibody showed strong specificity to the hydroxylated form (Fig. 1b , top two panels), in contrast to the equal recognition of both HIF-1 forms by the anti-HIF-1 antibody (bottom panel). Recognition of Hyp-564 by the anti-Hyp-564 was proportional to the increasing amount of hydroxylated HIF-1.

View larger version (45K): [in this window] [in a new window]   Figure 1. Leu-574 of HIF-1 is required for prolyl hydroxylation. a) HIF-1 and its proline mutants were transiently expressed in 293 cells. After 4 h Cbz-LLL treatment, their hydroxylation status was determined by Western blot with an antibody against Hyp-564 (-Hyp-564, top) and their expression levels were analyzed by an antibody against HIF-1 (-HIF-1, bottom). b) Nonhydroxylated and hydroxylated HIF-1 were in vitro translated in the presence of DFO or FeCl2. The translated products were loaded at increasing amounts (0.17, 0.33, and 0.5 µL) and subjected to Western blot with anti-Hyp-564 (top two panels) and anti-HIF-1 (bottom) antibodies. Autoradiography of different exposure times (in seconds) is indicated. c, d) HIF-1 and L574S mutant were evaluated for prolyl hydroxylation under normoxic and DFO-treated conditions with antibodies (c). Signal intensity is specified at the bottom of each lane. Ratio of HIF-1 containing Hyp-564 to a total amount of HIF-1 (Hyp-564/HIF-1), as determined by densitometry, is shown in a bar graph with means + SE from 3 independent experiments (d).

Consistent with the fact that HIF-1 is hydroxylated in normoxia, the anti-Hyp-564 detected the hydroxylated form primarily in normoxic cell extracts (Fig. 1c , top panel). The anti-HIF-1 antibody showed HIF-1 expression predominantly in hypoxic cell extracts (bottom panel). In keeping with the hypothesis that Leu-574 is involved in prolyl hydroxylation, mutation of Leu-574 markedly reduced hydroxylated HIF-1 levels in normoxia. To determine the hydroxylated form in reference to the total amount of HIF-1, we quantified the signals by densitometry and presented the ratio of the two in arbitrary numbers (Fig. 1d ). In comparison to normoxia, DFO treatment reduced prolyl hydroxylation by 90% in wild-type HIF-1. A similar reduction of the ratio was observed with the L574S mutant expressed in normoxia; DFO treatment resulted in an additional decrease. These results provide evidence that Leu-574 is involved in Pro-564 hydroxylation. To corroborate this finding, we compared the hydroxylation status of wild-type HIF-1 with the L574S mutant in the presence of Cbz-LLL. The ratio of hydroxylated Pro-564 of Leu-574 mutant under normoxia was only one-fifth that of the wild-type HIF-1. These data support that mutation of Leu-574 affects Pro-564 hydroxylation.

We analyzed the role of Leu-574 in prolyl hydroxylation with C-ODD (in the absence of Pro-402). C-ODD and its L574S mutant in Gal4 fusions were transfected in 293 cells and assayed for prolyl hydroxylation. Whereas DFO significantly diminished the ratio of hydroxylated form of Gal4-C-ODD, mutation of Leu-574 further decreased the ratio in normoxia and iron chelation. We previously showed that deletion of C-ODD from the carboxyl-terminal (amino acids 574-603) rendered the protein more stable than mutation of Leu-574 alone, suggesting involvement of the deleted region in protein degradation. As expected, such a deletion mutant (amino acids 498-573) ablated hydroxylation, in contrast to another deletion mutant (amino acids 498-574) that contains Leu-574. We conclude that Leu-574 of HIF-1 is essential for prolyl hydroxylation; residues 575-603 may also participate in the process.

3. Leu-574 of HIF-1 is required for PHD2/HPH2 binding
The role of Leu-574 in prolyl hydroxylation led to the question of whether the leucine is involved in the interaction with the HIF prolyl-4-hydroxylases that modify Pro-564. We performed coimmunoprecipitations of C-ODD with HIF prolyl hydroxylases. Figure 2 shows that in the presence of Cbz-LLL, PHD2/HPH2 was coprecipitated with C-ODD only when both were ectopically expressed in 293 cells (lanes 1, 3, 5). No coprecipitation was detected when Leu-574 was mutated, indicating a requirement of Leu-574 for PHD2/HPH2 interaction. The higher levels of Gal4 fusions in the absence ectopically expressed PHD2/HPH2 (top and middle panels) support the specificity of coimmunoprecipitated PHD2/HPH2. We also examined other possible interactions of C-ODD with PHD1/HPH3 or PHD3/HPH1 but failed to detect similar interaction under the same conditions. These data indicate that Leu-574 participates in the recruitment of PHD2/HPH2 for the subsequent hydroxylation of Pro-564.

View larger version (75K): [in this window] [in a new window]   Figure 2. Leu-574 of HIF-1 is required for PHD2/HPH2 binding. Gal4-C-ODD and its L574S mutant were transfected into 293 cells in the presence or absence of V5-tagged PHD2/HPH2. After Cbz-LLL treatment, C-ODD-PHD2/HPH2 interaction was revealed by coimmunoprecipitation with anti-Gal4 antibodies, followed by Western blot with anti-V5 antibodies (bottom). Expression levels of Gal4 fusions and V5-tagged PHD2/HPH2 were determined (upper and middle panels).

CONCLUSIONS AND SIGNIFICANCE

We show here that in human HIF-1, Leu-574, 10 residues downstream of Pro-564, is required for PHD2/HPH2 binding and prolyl hydroxylation. HIF-1 Leu-574 is the first identified residue distal from Pro-564 but crucial for prolyl hydroxylation. Although the nine-residue spacing between Pro-564 and Leu-574 is not obligatory, the degree of Pro-564 hydroxylation appears to be enhanced by alteration of the spacing. Asn-803 of HIF-1, another target of hydroxylation by an asparaginyl hydroxylase FIH-1, shares the same spacing as Pro-564 with Leu-813, shown to be involved in FIH-1 interaction. These findings imply that the distal leucines are generally required for hydroxylase binding. It would be of great interest to test the role of conserved Leu-411 in Pro-402 hydroxylation.

Our evidence shows that Leu-574 participates in the interaction with one of the HIF prolyl hydroxylases, PHD2/HPH2; mutation of the leucine resulted in loss of binding to the hydroxylase, suggesting a requirement for Leu-574 in recruiting the hydroxylase. The PHD2/HPH2 interaction with HIF-1 in vivo is consistent with its highest enzymatic activity among the three HIF prolyl hydroxylases for HIF-1 hydroxylation in vitro. That PHD2/HPH2 is the key prolyl hydroxylase for controlling normoxic degradation of HIF-1 further supports an essential role of Leu-574 in Pro-564 hydroxylation (Fig. 3 ). Only HIF-1 Leu-574 has been shown to play a consistent role in PHD2/HPH2 recruitment, prolyl hydroxylation, and consequently VHL binding. Although Leu-574 does not play a regulatory role in prolyl hydroxylation, its structural requirement may serve as a reliable site for drug targeting of HIF-1 activity, thereby altering the cellular response to hypoxia.

View larger version (17K): [in this window] [in a new window]   Figure 3. Key residues involved in HIF-1 degradation. Sequence events of HIF-1 degradation are schematically depicted. Leu-574 of HIF-1 is required for recruitment of HIF prolyl hydroxylase PHD2/HPH2, resulting in prolyl hydroxylation, VHL binding, and ultimately ubiquitylation (Lys-Ub) and degradation of HIF-1.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1233fje;

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