The hypoxia-inducible factors (HIFs) 1alpha and 2alpha are key mammalian transcription factors that exhibit dramatic increases in both protein stability and intrinsic transcriptional potency during low-oxygen stress. This increased stability is due to the absence of proline hydroxylation, which in normoxia promotes binding of HIF to the von Hippel-Lindau (VHL tumor suppressor) ubiquitin ligase. We now show that hypoxic induction of the COOH-terminal transactivation domain (CAD) of HIF occurs through abrogation of hydroxylation of a conserved asparagine in the CAD. Inhibitors of Fe(II)- and 2-oxoglutarate-dependent dioxygenases prevented hydroxylation of the Asn, thus allowing the CAD to interact with the p300 transcription coactivator. Replacement of the conserved Asn by Ala resulted in constitutive p300 interaction and strong transcriptional activity. Full induction of HIF-1alpha and -2alpha, therefore, relies on the abrogation of both Pro and Asn hydroxylation, which during normoxia occur at the degradation and COOH-terminal transactivation domains, respectively.

Mammalian cells adapt to hypoxic conditions through a transcriptional response pathway mediated by the hypoxia-inducible factor, HIF. HIF transcriptional activity is suppressed under normoxic conditions by hydroxylation of an asparagine residue within its C-terminal transactivation domain, blocking association with coactivators.Here we show that the protein FIH-1, previously shown to interact with HIF, is an asparaginyl hydroxylase. Like known hydroxylase enzymes, FIH-1 is an Fe(II)-dependent enzyme that uses molecular O 2 to modify its substrate. Together with the recently discovered prolyl hydroxylases that regulate HIF stability, this class of oxygen-dependent enzymes comprises critical regulatory components of the hypoxic response pathway. Received March 14, 2002; revised version accepted April 30, 2002. Almost all mammalian cells possess the ability to recognize changes in the local availability of oxygen. When oxygen levels are low (hypoxia), a conserved hypoxic response pathway is activated. At the center of this pathway lies the ubiquitously expressed transcription factor hypoxia-inducible factor (HIF) (Semenza 1999). HIF is a heterodimer composed of an alpha subunit, HIF-1␣ or the paralogs HIF-2␣ or HIF-3␣ (Tian et al. 1997;Gu et al. 1998;O'Rourke et al. 1999;Srinivas et al. 1999), and the HIF-1␤ subunit, also known as the aryl hydrocarbon receptor nuclear translocator (ARNT) (Wang et al. 1995). Whereas HIF-1␤ expression and activity levels remain largely unaffected by changes in oxygen levels, the HIF-␣ subunit is strongly induced following exposure to hypoxic conditions. Two primary mechanisms by which HIF-␣ activity is regulated by oxygen have been identified. Under normoxic conditions, the oxygen-dependent degradation domain (ODD) within the HIF-␣ subunit is recognized by the product of the von-Hippel Lindau tumor suppressor gene (pVHL) (Maxwell et al. 1999). pVHL is a component of a protein-ubiquitin ligase complex that targets the alpha subunit for degradation by the proteasome (Maxwell et al. 1999;Cockman et al. 2000;Ohh et al. 2000;Tanimoto et al. 2000). pVHL recognition of HIF-␣ is dependent on hydroxylation of proline residues within the ODD (Ivan et al. 2001;Jaakkola et al. 2001;Yu et al. 2001). Under hypoxic conditions, prolyl hydroxylation is blocked, resulting in increased HIF-␣ stability and accumulation (Ivan et al. 2001;Jaakkola et al. 2001;Yu et al. 2001). This posttranslational modification is carried out by a family of prolyl hydroxylase enzymes that bear structural and functional similarities to previously characterized hydroxylases (Bruick and McKnight 2001;Epstein et al. 2001). Like these enzymes, the HIF prolyl hydroxylase enzymes use Fe(II) to bind O 2 to hydroxylate both 2-oxoglutarate and the target proline residue (Bruick and McKnight 2001;Epstein et al. 2001). Because these enzymes bind oxygen directly, it has been speculated that they may be critical oxygen sensors within the hypoxic response pathway.In addition to inducing HIF stability, hypoxic conditions promote the ...

Mammalian spermatozoa must undergo epididymal maturation in the male reproductive tract and capacitation in the female tract before acquiring the ability to fertilize an oocyte. Previous studies from our laboratory have demonstrated a causal relationship between capacitation-associated surface phosphotyrosine expression and the ability of mouse spermatozoa to recognize the oocyte and engage in sperm-zona pellucida interaction. Our previous analyses of the surface phosphoproteome of capacitated murine spermatozoa identified two molecular chaperones, heat shock protein (HSP) D1 and HSP90B1, with well-characterized roles in protein folding and the assemblage of multimeric protein complexes. The expression of these chaperones was restricted to the rostral aspect of the sperm head, in an ideal position to mediate sperm-zona pellucida interaction. Herein, we report the characterization of an additional chaperone in this location, HSPE1 (chaperonin 10; HSP10). This chaperone was identified using a coimmunoprecipitation strategy employing HSPD1 as bait. The putative interaction between HSPE1 and HSPD1 was supported by reciprocal immunoprecipitation and colocalization studies, which demonstrated the coordinated appearance of both proteins on the surface of the sperm head during capacitation. However, the surface exposure of the protein was lost upon induction of acrosomal exocytosis, as would be expected of a protein potentially involved in sperm-zona pellucida interaction. Collectively, these data invite speculation that a number of molecular chaperones are involved in modification of the sperm surface during capacitation to render these cells functionally competent to engage the process of fertilization.