Asparagine Hydroxylation of the HIF Transactivation Domain: A Hypoxic Switch
Tóm tắt
The hypoxia-inducible factors (HIFs) 1α and 2α 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-1α and -2α, therefore, relies on the abrogation of both Pro and Asn hydroxylation, which during normoxia occur at the degradation and COOH-terminal transactivation domains, respectively.
Từ khóa
Tài liệu tham khảo
J. F. O'Rourke
Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr.
Supplementary material is available on Science Online at www.sciencemag.org/cgi/content/full/295/5556/858/DC1
D. Lando et al. data not shown.
For exposure to hypoxia (<1% O 2 ) cells were placed inside an airtight chamber along with an AnaeroGen sachet (OXOID Hampshire UK) according to the manufacturer's instructions.
To generate the stable cell line we subcloned cDNA encoding HisMycHIF-2α into the pEFIRES-puro vector (27) and transfected human embryonic kidney HEK 293T cells. Cells were pooled after 2 weeks of selection with 1 μg/ml puromycin. A control cell line was derived from the same selection process after transfection with the empty pEFIRES-puro vector.
Cells were lysed with binding buffer [100 mM Na-phosphate (pH 8.0) 8 M urea 0.1% NP40 0.15 M NaCl 2 5 mM imidazole with protease and phosphatase inhibitors]. Clarified lysate was incubated with Ni-IDA agarose (Scientifix Australia) and after extensive washing eluted with 100 mM Na-phosphate (pH 8.0) 8 M urea and 200 mM imidazole. Eluted protein was loaded onto a butyl C4 high-performance liquid chromatography (HPLC) column (Brownlee PerkinElmer) that had been equilibrated in 0.1% (v/v) trifluoroacetic acid (TFA). HIF-2α774-874 was then eluted with an increasing gradient of 80% (v/v) acetonitrile/0.1% (v/v) TFA.
An anaerobic workstation MkIII (Don Whitley Scientific UK) was used to prepare hypoxic HIF-2α774–874 protein for MS analysis. Cells were washed and lysed inside the anaerobic workstation with buffers that had been deoxygenated overnight. After cell lysis the purification was carried out at ambient O 2 conditions.
The HPLC fractions were reduced and alkylated with iodoacetamide before trypsin digestion. Theoretical products of tryptic digestion were generated by using the Bioanalyst component of Analyst QS software (Applied Biosystems). Digests were analyzed by using a Bruker Reflex MALDI-TOF-MS operated in the positive ion reflector mode and data were acquired and analyzed with the Bruker XMass suite of software. Post-source decay (PSD) analysis was performed using the Bruker Reflex mass spectrometer (28).
Peptides were subjected to partial sequence analysis by MS/MS in the positive ion mode with a Sciex QSTAR-Pulsar Qq-TOF-MS under the control of Analyst QS software. Tryptic digests were sprayed from 60% (v/v) aqueous methanol containing 0.1% (v/v) formic acid. Diluted digests (∼2 μl) were loaded into drawn capillaries coated with gold or platinum (Protana NanoES capillaries) and fitted onto a Protana NanoES electrospray ion source. Ions were sprayed with a potential of 850 V on the sample capillary. Collisionally activated decomposition of peptides was achieved by selecting doubly charged ions of interest using Q1 at low resolution and manually varying the energy of collisions with nitrogen gas to achieve optimal spread of fragments across the desired TOF mass ranges (29). Identification of proteins was achieved by using MS/MS spectra to search the Mascot database (www.matrixscience.com) with mass error constraints of 0.1 and 0.05 daltons for parent ions and fragments ions respectively.
The GST-p300CH1 domain (amino acids 300 to 528) fusion protein was expressed from pGEX-4T3/p300CH1 and purified from bacterial extract by glutathione agarose. A whole-cell extract (200 μg) from transfected cells was mixed with 2 μg of glutathione bound GST-CH1 in binding buffer [20 mM tris-HCl (pH 8.0) 150 mM NaCl 0.1% NP40 1 mM dithiothreitol 2 μM EDTA 20 μM ZnCl 2 and protease inhibitors] and incubated for 2 hours at 4°C with gentle rotation. The beads were pelleted then washed three times with binding buffer and bound proteins were eluted for SDS–polyacrylamide gel electrophoresis (SDS-PAGE) with 1× SDS sample buffer.
We thank S. Pyke for help with the synthesis of DMOG P. Ratcliffe for the gift of KA13.5 cells Y. Fujii-Kuriyama for murine HIF-2α cDNA and the HRE reporter gene and S. McKnight for critical reading of the manuscript.