Independent function of two destruction domains in hypoxia‐inducible factor‐α chains activated by prolyl hydroxylation

Oxygen‐dependent proteolytic destruction of hypoxia‐inducible factor‐α (HIF‐α) subunits plays a central role in regulating transcriptional responses to hypoxia. Recent studies have defined a key function for the von Hippel–Lindau tumour suppressor E3 ubiquitin ligase (VHLE3) in this process, and have defined an interaction with HIF‐1α that is regulated by prolyl hydroxylation. Here we show that two independent regions within the HIF‐α oxygen‐dependent degradation domain (ODDD) are targeted for ubiquitylation by VHLE3 in a manner dependent upon prolyl hydroxylation. In a series of in vitro and in vivo assays, we demonstrate the independent and non‐redundant operation of each site in regulation of the HIF system. Both sites contain a common core motif, but differ both in overall sequence and in the conditions under which they bind to the VHLE3 ligase complex. The definition of two independent destruction domains implicates a more complex system of pVHL–HIF‐α interactions, but reinforces the role of prolyl hydroxylation as an oxygen‐dependent destruction signal.

[1]  J. Pouysségur,et al.  p42/p44 Mitogen-activated Protein Kinases Phosphorylate Hypoxia-inducible Factor 1α (HIF-1α) and Enhance the Transcriptional Activity of HIF-1* , 1999, The Journal of Biological Chemistry.

[2]  C. Wykoff,et al.  The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis , 1999, Nature.

[3]  S. McKnight,et al.  Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells. , 1997, Genes & development.

[4]  Michael I. Wilson,et al.  Targeting of HIF-α to the von Hippel-Lindau Ubiquitylation Complex by O2-Regulated Prolyl Hydroxylation , 2001, Science.

[5]  R. Conaway,et al.  Activation of HIF1alpha ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[6]  M. Ivan,et al.  HIFα Targeted for VHL-Mediated Destruction by Proline Hydroxylation: Implications for O2 Sensing , 2001, Science.

[7]  T. Dingermann.  [Do cells sense oxygen?]. , 2001, Pharmazie in unserer Zeit.

[8]  J. Rosenbloom,et al.  Incorporation of 3,4-dehydroproline into protocollagen and collagen. Limited hydroxylation of proline and lysine in the same polypeptide. , 1970, The Journal of biological chemistry.

[9]  S. Bhattacharya,et al.  Functional role of p35srj, a novel p300/CBP binding protein, during transactivation by HIF-1. , 1999, Genes & development.

[10]  G. Semenza,et al.  HIF-1 and human disease: one highly involved factor. , 2000, Genes & development.

[11]  G. Semenza,et al.  Transactivation and Inhibitory Domains of Hypoxia-inducible Factor 1α , 1997, The Journal of Biological Chemistry.

[12]  L. Poellinger,et al.  Signal transduction in hypoxic cells: inducible nuclear translocation and recruitment of theCBP/p300 coactivator by the hypoxia‐induciblefactor‐1α , 1998, The EMBO journal.

[13]  G. Semenza,et al.  Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O 2 tension , 2002 .

[14]  Peter Jackson,et al.  The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases. , 2000, Trends in cell biology.

[15]  J. Caro,et al.  Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. , 1997, The Journal of biological chemistry.

[16]  Selection and Analysis of a Mutant Cell Line Defective in the Hypoxia-inducible Factor-1 a-Subunit (HIF-1a) , 1998 .

[17]  G. Semenza,et al.  The human hypoxia-inducible factor 1alpha gene: HIF1A structure and evolutionary conservation. , 1998, Genomics.

[18]  G. Semenza,et al.  Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1alpha. , 2000, Genes & development.

[19]  J. Pouysségur,et al.  Signaling angiogenesis via p42/p44 MAP kinase and hypoxia. , 2000, Biochemical pharmacology.

[20]  Y. Fujii‐Kuriyama,et al.  Molecular mechanisms of transcription activation by HLF and HIF1α in response to hypoxia: their stabilization and redox signal‐induced interaction with CBP/p300 , 1999, The EMBO journal.

[21]  G. Semenza,et al.  Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension. , 1996, The American journal of physiology.

[22]  M. Gassmann,et al.  Induction of HIF–1α in response to hypoxia is instantaneous , 2001, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[23]  J. Caro,et al.  Hypoxia-inducible Factor 1α (HIF-1α) Protein Is Rapidly Degraded by the Ubiquitin-Proteasome System under Normoxic Conditions , 1997, The Journal of Biological Chemistry.

[24]  G. Semenza,et al.  Hypoxia-inducible factor 1alpha protein expression is controlled by oxygen-regulated ubiquitination that is disrupted by deletions and missense mutations. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[25]  M. Tyers,et al.  Combinatorial control in ubiquitin-dependent proteolysis: don't Skp the F-box hypothesis. , 1998, Trends in genetics : TIG.

[26]  R. Wenger.  Mammalian oxygen sensing, signalling and gene regulation. , 2000, The Journal of experimental biology.

[27]  P. Ratcliffe,et al.  Oxygen-regulated and Transactivating Domains in Endothelial PAS Protein 1: Comparison with Hypoxia-inducible Factor-1α* , 1999, The Journal of Biological Chemistry.

[28]  L. Huang,et al.  Regulation of hypoxia-inducible factor 1α is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway , 1998 .

[29]  S. White,et al.  Dynamic, site-specific interaction of hypoxia-inducible factor-1alpha with the von Hippel-Lindau tumor suppressor protein. , 2001, Cancer research.

[30]  G. Semenza.  Perspectives on Oxygen Sensing , 1999, Cell.

[31]  M. Nagao,et al.  Activation of Hypoxia-inducible Factor-1; Definition of Regulatory Domains within the α Subunit* , 1997, The Journal of Biological Chemistry.

[32]  K. Kivirikko,et al.  Prolyl 4-hydroxylases and their protein disulfide isomerase subunit. , 1998, Matrix biology : journal of the International Society for Matrix Biology.

[33]  L. Poellinger,et al.  Mechanism of regulation of the hypoxia‐inducible factor‐1α by the von Hippel‐Lindau tumor suppressor protein , 2000, The EMBO journal.

[34]  Eamonn R. Maher,et al.  Hypoxia Inducible Factor-α Binding and Ubiquitylation by the von Hippel-Lindau Tumor Suppressor Protein* , 2000, The Journal of Biological Chemistry.

[35]  M. Ivan,et al.  Ubiquitination of hypoxia-inducible factor requires direct binding to the β-domain of the von Hippel–Lindau protein , 2000, Nature Cell Biology.

[36]  M. Gstaiger,et al.  The von Hippel-Lindau tumor suppressor protein is a component of an E3 ubiquitin-protein ligase activity. , 1999, Genes & development.

[37]  W. Kaelin,et al.  Structure of the VHL-ElonginC-ElonginB complex: implications for VHL tumor suppressor function. , 1999, Science.

[38]  D. Livingston,et al.  Activation of Hypoxia-inducible Transcription Factor Depends Primarily upon Redox-sensitive Stabilization of Its α Subunit* , 1996, The Journal of Biological Chemistry.

[39]  P. Ratcliffe,et al.  Selection and analysis of a mutant cell line defective in the hypoxia-inducible factor-1 alpha-subunit (HIF-1alpha). Characterization of hif-1alpha-dependent and -independent hypoxia-inducible gene expression. , 1998, The Journal of biological chemistry.