Hopfer Ulrike, Hopfer Helmut, Jablonski Katarina, Stahl Rolf A K, Wolf Gunter
Department of Medicine, University of Hamburg, Martinistr. 52, D-20246 Hamburg.
J Biol Chem. 2006 Mar 31;281(13):8645-55. doi: 10.1074/jbc.M513751200. Epub 2006 Jan 3.
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.
缺氧诱导因子-1（HIF-1）是一种由氧敏感的α亚基和β亚基组成的转录复合物，在细胞适应低氧环境中起关键作用。在常氧条件下，HIF-1的α亚基被脯氨酰羟化酶（PHD）家族羟基化，随后被靶向蛋白酶体降解。已鉴定出三种不同的PHD，但它们在体内作用的差异仍不清楚。PHD3在缺氧时显著表达，具有高底物特异性，并且已在其他信号通路中被鉴定出来。因此，PHD3可能羟基化不同的底物和/或将不同的细胞反应与HIF联系起来。我们通过酵母双杂交实验筛选cDNA文库，鉴定出一种新的WD重复蛋白，最近被命名为Morg1（丝裂原活化蛋白激酶组织者1）。PHD3与Morg1之间的相互作用在体外和体内均得到证实。通过克隆Morg1的全长cDNA，我们发现了七个WD重复结构域。通过共聚焦显微镜观察，两种蛋白在细胞质和细胞核内共定位，并且在Northern印迹中显示出相似的组织表达模式。结合发生在预测位于一个螺旋桨叶片顶表面的保守区域。最后，Morg1可降低HIF介导的报告基因活性，当Morg1与PHD3共表达时，活性降低至基础水平。通过RNA干扰抑制Morg1或PHD3会导致HIF-1活性显著增加。这些结果表明，Morg1最有可能通过作为分子支架与PHD3特异性相互作用。这种相互作用可能在HIF调节和其他信号通路之间提供一个分子框架。
