Abstract
The hypoxia-inducible factor-1alpha (HIF-1alpha) is a master regulator of the cellular response to decreased oxygen levels. This transcription factor is highly unstable at normal oxygen concentrations and is rapidly stabilized by hypoxia. At normoxia two specific proline residues (Pro(402) and Pro(563)) of mHIF-1alpha are hydroxylated and recognized by the von Hippel-Lindau E3 ubiquitin ligase (pVHL) complex, which upon binding mediates degradation of the protein. Previous studies have demonstrated that these two proline residues are critical for high affinity binding to pVHL. We have performed a detailed analysis of a mutant form of HIF-1alpha, where both these proline residues have been mutated, and we have uncovered a novel degradation pathway, to which the HIF-1alpha mutant protein is not resistant. Our results show that the HIF-1alpha double proline mutant undergoes ubiquitination and proteasome-dependent degradation, and retains the ability to be stabilized in response to hypoxia and CoCl(2) treatment. However in contrast to the wild-type protein, stabilization of the mutant was only observed within short periods of hypoxia exposure (1-2 h). Degradation assays in the presence of the expressed prolyl hydroxylases (PHDs) 1-3 showed that, unlike the wild-type protein, the HIF-1alpha mutant was resistant to these hydroxylases. However, experiments knocking-down expression of pVHL by RNA interference showed that the HIF-1alpha mutant is degraded and ubiquitinated by a pVHL-mediated mechanism. In conclusion, we show the first evidence of a novel mechanism of degradation of HIF-1alpha at normoxia that involves pVHL but is not mediated by PHDs 1-3 or by degradation boxes surrounding Pro(402) and Pro(563).
Highlights
hypoxia-inducible factor-1 (HIF-1) target genes encode proteins critical for anaerobic metabolism, angiogenesis, and erythropoiesis
Proteins were visualized using enhanced chemiluminescence (Amersham Biosciences). Mutation of Both Pro402 and Pro563 Does Not Render HIF-1␣ Protein Stable at Normoxia—Hydroxylation of specific proline residues has been shown to mediate recognition of HIF-1␣ by pVHL. pVHL belongs to a multiprotein complex with E3 ubiquitin ligase activity that mediates ubiquitination of HIF-1␣ leading to its proteasome-dependent degradation
Cells were treated with CoCl2, an iron antagonist that is known to act as an hypoxia-mimicking agent because Fe(II) is a critical cofactor required for the reactions performed by the different hydroxylases that regulate HIF-1␣ activity [1, 2]
Summary
Plasmid Constructs—pFLAG-PHD1 and pFLAG-PHD3 were cloned from cDNAs generated by RT-PCR using RNA from HeLa cells. 25 g of whole cell extract proteins were separated by SDS-PAGE and analyzed by immunoblotting. Protein G-Sepharose beads were blocked with 1% bovine serum albumin in TBS (150 mM NaCl, 50 mM Tris-Cl, pH 7.4) before immunized with anti-FLAG antibodies (Sigma). Eluates and 25 g of whole cell extract proteins were separated by SDS-PAGE and analyzed by immunoblotting. For precipitation of ubiquitinated-HIF-1␣ proteins, 5 l of anti-FLAG antibody was incubated with 25 l of protein G-Sepharose beads after blocking with 1% bovine serum albu-. For precipitation of HIF-1␣ protein/pVHL complexes, 5 l of antiFLAG antibody was incubated with 25 l of protein G-Sepharose beads after blocking with 1% bovine serum albumin, and followed by incubation with 1.5 mg of whole cell extracts overnight at 4 °C.
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