Abstract
Hypoxia-inducible factor-1 (HIF-1), a transcriptional complex composed of an oxygen-sensitive alpha- and a beta-subunit, plays a pivotal role in cellular adaptation to low oxygen availability. Under normoxia, the alpha-subunit of HIF-1 is hydroxylated by a family of prolyl hydroxylases (PHDs) and consequently targeted for proteasomal degradation. Three different PHDs have been identified, but the difference among their in vivo roles remain unclear. PHD3 is strikingly expressed by hypoxia, displays high substrate specificity, and has been identified in other signaling pathways. PHD3 may therefore hydroxylate divergent substrates and/or connect divergent cellular responses with HIF. We identified a novel WD-repeat protein, recently designated Morg1 (MAPK organizer 1), by screening a cDNA library with yeast two-hybrid assays. The interaction between PHD3 and Morg1 was confirmed in vitro and in vivo. We found seven WD-repeat domains by cloning the full-length cDNA of Morg1. By confocal microscopy both proteins co-localize within the cytoplasm and the nucleus and display a similar tissue expression pattern in Northern blots. Binding occurs at a conserved region predicted to the top surface of one propeller blade. Finally, HIF-mediated reporter gene activity is decreased by Morg1 and reduced to basal levels when Morg1 is co-expressed with PHD3. Suppression of Morg1 or PHD3 by stealth RNA leads to a marked increase of HIF-1 activity. These results indicate that Morg1 specifically interacts with PHD3 most likely by acting as a molecular scaffold. This interaction may provide a molecular framework between HIF regulation and other signaling pathways.
Highlights
Identified in human (HIF-1␣, -2␣, and -3␣) and a  subunit (HIF-1/ ARNT) [5,6,7]
Three positive clones encoded the carboxyl-terminal half of Morg1 (Fig. 1, A and B), a WD-repeat protein recently described as a component of the ERK cascade [45]
The activity of the transcriptional complex of Hypoxia-inducible factor-1 (HIF-1)␣ is regulated by oxygen-dependent post-translational modifications that are mediated by specific 2-oxoglutarate and Fe(II)-dependent dioxygenases (PHDs)
Summary
All chemicals other than indicated were purchased from Sigma, restriction and modifying enzymes from New England Biolabs, the yeast two-hybrid system vectors, primers, yeast strain, yeast plasmid purification products, cDNA library, and co-immunoprecipitation products were from Clontech. Yeast Two-hybrid Screening pGBKT7 was expressed as a fusion protein to a GAL4 DNA binding domain and a c-Myc epitope tag using tryptophan as selection marker. A rat brain cDNA library cloned into pACT2 was expressed as a fusion protein to the GAL4 activation domain and a HA epitope tag using leucine as selection marker. Transformants were screened on SD-agar plates lacking adenine, histidine, leucine, tryptophan, and containing X-␣-galactosidase (high stringency selection). To eliminate false positive clones yeast plasmids were isolated using a yeast plasmid isolation KIT (Clontech), transformed into Escherichia coli, purified, retransformed into the yeast strain expressing PHD3, and streaked out twice. Positive clones from this screening were subjected to direct sequencing.
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