ABSTRACT
HIF-1alpha is the inducible subunit of the dimeric transcription factor HIF-1 (Hypoxia Inducible Factor 1). It is induced by hypoxia and hypoxia-mimetics in most cell types, as well as non-hypoxic signals such as growth factors, cytokines and oncogenes, often in a cell specific manner. HIF-1 is present in virtually all cells of higher eukaryotes and its function is of great biomedical relevance since it is highly involved in development, tumor progression and tissue ischemia. Intracellular signaling to HIF-1alpha, as well as its further action, involves its participation in numerous protein complexes. Using the yeast two-hybrid system we have identified MgcRacGAP (male germ cell Rac GTPase Activating Protein) as a HIF-1alpha interacting protein. The MgcRacGAP protein is a regulator of Rho proteins, which are principally involved in cytoskeletal organization. We have verified specific binding of HIF-1alpha and MgcRacGAP in vitro and in vivo in mammalian cells. We have additionally shown that MgcRacGAP overexpression inhibits HIF-1alpha transcriptional activity, without lowering HIF-1alpha protein levels, or altering its subcellular localization. Moreover, this inhibition is dependent on the MgcRacGAP domain that interacts with HIF-1alpha. In conclusion, our findings demonstrate that HIF-1alpha function is negatively affected by its interaction with MgcRacGAP.
Subject(s)
GTPase-Activating Proteins/metabolism , Gene Expression Regulation , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Transcription, Genetic , Animals , HeLa Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Luciferases/metabolism , Mice , Protein Binding , Protein Transport , Saccharomyces cerevisiae/cytology , Subcellular Fractions , Thermodynamics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Hypoxia inducible factor 1 (HIF-1), the master regulator of hypoxia-activated genes, is involved in many diseases and is a valid drug target. In order to develop a simple and genetically tractable in vivo system for HIF-1 analysis, we tested the inducible expression of both human HIF-1 subunits (HIF-1alpha and ARNT) in the yeast Saccharomyces cerevisiae and showed the formation of transcriptionally active HIF-1. The use of this system for the identification and characterization of HIF-1 effectors was first validated by showing that two chemical Hsp90 inhibitors, geldanamycin and radicicol, impaired the activity of HIF-1 in yeast. By applying this system in mutant yeast strains, we then identified Hsp90 co-chaperones, which were required for HIF-1 activity. Furthermore, using yeast strains co-expressing truncated forms of HIF-1alpha with ARNT or both HIF-1alpha and ARNT, we characterized fragments of HIF-1alpha that acted as dominant negative mutants and suppressed HIF-1 activity.
