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

Denise A. Chan, Patrick D. Sutphin, Shing Erh Yen, Amato J. Giaccia

Research output: Contribution to journalArticle

153 Citations (Scopus)

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-1α to the proteasome for degradation. Mutation of either proline is sufficient to partially stabilize HIF-1α under conditions of normoxia, but the specific contributions of each hydroxylation event to the regulation of HIF-1α are unknown. Here we show that the two ODDs of HIF-1α have independent yet interactive roles in the regulation of HIF-1α 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-1α 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-1α regulation under conditions of hypoxia than under conditions of normoxia. Together, these findings demonstrate that each hydroxylated proline of HIF-1α has a distinct activity in controlling HIF-1α stability in response to different levels of oxygenation.

Original languageEnglish (US)
Pages (from-to)6415-6426
Number of pages12
JournalMolecular and Cellular Biology
Volume25
Issue number15
DOIs
StatePublished - Aug 2005

Fingerprint

Hypoxia-Inducible Factor 1
Proline
Oxygen
Hydroxylation
Mutation
Proteolysis
Prolyl Hydroxylases
Ubiquitin-Protein Ligases
Proteasome Endopeptidase Complex

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Coordinate regulation of the oxygen-dependent degradation domains of hypoxia-inducible factor 1α. / Chan, Denise A.; Sutphin, Patrick D.; Yen, Shing Erh; Giaccia, Amato J.

In: Molecular and Cellular Biology, Vol. 25, No. 15, 08.2005, p. 6415-6426.

Research output: Contribution to journalArticle

Chan, Denise A. ; Sutphin, Patrick D. ; Yen, Shing Erh ; Giaccia, Amato J. / Coordinate regulation of the oxygen-dependent degradation domains of hypoxia-inducible factor 1α. In: Molecular and Cellular Biology. 2005 ; Vol. 25, No. 15. pp. 6415-6426.
@article{cc89255196a746468565abb388a76abe,
title = "Coordinate regulation of the oxygen-dependent degradation domains of hypoxia-inducible factor 1α",
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-1α to the proteasome for degradation. Mutation of either proline is sufficient to partially stabilize HIF-1α under conditions of normoxia, but the specific contributions of each hydroxylation event to the regulation of HIF-1α are unknown. Here we show that the two ODDs of HIF-1α have independent yet interactive roles in the regulation of HIF-1α 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-1α 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-1α regulation under conditions of hypoxia than under conditions of normoxia. Together, these findings demonstrate that each hydroxylated proline of HIF-1α has a distinct activity in controlling HIF-1α stability in response to different levels of oxygenation.",
author = "Chan, {Denise A.} and Sutphin, {Patrick D.} and Yen, {Shing Erh} and Giaccia, {Amato J.}",
year = "2005",
month = "8",
doi = "10.1128/MCB.25.15.6415-6426.2005",
language = "English (US)",
volume = "25",
pages = "6415--6426",
journal = "Molecular and Cellular Biology",
issn = "0270-7306",
publisher = "American Society for Microbiology",
number = "15",

}

TY - JOUR

T1 - Coordinate regulation of the oxygen-dependent degradation domains of hypoxia-inducible factor 1α

AU - Chan, Denise A.

AU - Sutphin, Patrick D.

AU - Yen, Shing Erh

AU - Giaccia, Amato J.

PY - 2005/8

Y1 - 2005/8

N2 - 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-1α to the proteasome for degradation. Mutation of either proline is sufficient to partially stabilize HIF-1α under conditions of normoxia, but the specific contributions of each hydroxylation event to the regulation of HIF-1α are unknown. Here we show that the two ODDs of HIF-1α have independent yet interactive roles in the regulation of HIF-1α 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-1α 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-1α regulation under conditions of hypoxia than under conditions of normoxia. Together, these findings demonstrate that each hydroxylated proline of HIF-1α has a distinct activity in controlling HIF-1α stability in response to different levels of oxygenation.

AB - 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-1α to the proteasome for degradation. Mutation of either proline is sufficient to partially stabilize HIF-1α under conditions of normoxia, but the specific contributions of each hydroxylation event to the regulation of HIF-1α are unknown. Here we show that the two ODDs of HIF-1α have independent yet interactive roles in the regulation of HIF-1α 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-1α 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-1α regulation under conditions of hypoxia than under conditions of normoxia. Together, these findings demonstrate that each hydroxylated proline of HIF-1α has a distinct activity in controlling HIF-1α stability in response to different levels of oxygenation.

UR - http://www.scopus.com/inward/record.url?scp=22544464403&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=22544464403&partnerID=8YFLogxK

U2 - 10.1128/MCB.25.15.6415-6426.2005

DO - 10.1128/MCB.25.15.6415-6426.2005

M3 - Article

VL - 25

SP - 6415

EP - 6426

JO - Molecular and Cellular Biology

JF - Molecular and Cellular Biology

SN - 0270-7306

IS - 15

ER -