MgcRacGAP Interacts with HIF-1α and Regulates its Transcriptional Activity

Abstract

HIF-1α 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-1α, 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-1α 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-1α and MgcRacGAP in vitro and in vivo in mammalian cells. We have additionally shown that MgcRacGAP overexpression inhibits HIF-1α transcriptional activity, without lowering HIF-1α protein levels, or altering its subcellular localization. Moreover, this inhibition is dependent on the MgcRacGAP domain that interacts with HIF-1α. In conclusion, our findings demonstrate that HIF-1α function is negatively affected by its interaction with MgcRacGAP.

Mortalin-mediated and ERK-controlled targeting of HIF-1α to mitochondria confers resistance to apoptosis under hypoxia

“…To generate wild-type and mutant pEGFP-HIF-1α-575-826 forms, cDNA was obtained by PCR using the corresponding pEGFP-HIF-1α forms as a template, and sub-cloned as BamHI fragments into pEGFP-C1. The following antibodies were used: affinity-purified rabbit polyclonal antibody against HIF-1α (1:1000; Lyberopoulou et al, 2007); rabbit polyclonal antibodies against mortalin (sc-13967, 1:1000 dilution), TOM20 (sc-11415, 1:1000 dilution) and Gal4-DBD (sc-577, 1:500 dilution), mouse monoclonal antibody against C-terminus of PARP-1 (sc-8007, 1:5000 dilution) or goat polyclonal antibody against VDAC1 (sc-8828, 1:500 dilution) all from Santa Cruz Biotechnology (Dallas, TX); rabbit polyclonal antibodies against phospho-ERK1/2 (9101, 1:1000 dilution), ERK1/2 (9102, 1:1000 dilution), HSP60 (4870, 1:1000 dilution), caspase 3 (9662, 1:1000 dilution) and cytochrome c (4272, 1:500 dilution), rabbit monoclonal antibodies against HK-II (6867, 1:1000 dilution) and cleaved caspase-3 (9664, 1:500 dilution) and mouse monoclonal antibody against actin (3700, 1:5000 dilution) all from Cell Signaling (Danvers, MA); mouse monoclonal antibodies against HIF-1α (610959, 1:1000 dilution), ARNT (611079, 1:500 dilution) and phosphoserine (612547, 1:1000 dilution) from BD Biosciences (San Jose, CA); mouse monoclonal antibody against the Nterminus of PARP-1 (ALX-804-211, 1:5000 dilution) from Enzo Life Sciences (Farmingdale, NY); and rabbit polyclonal antibody against HIF-2α (ΝΒ100-122, 1:1000 dilution) from Novus Europe (Cambridge, UK) or against Flag (F4042, 1:10,000 dilution) from Sigma-Aldrich (St Louis, MO). Immunoblotting was performed as previously described (Mylonis et al, 2006).…”

Hypoxia causes triglyceride accumulation via HIF-1-mediated stimulation of lipin 1 expression

“…Antibodies used for western blotting, immunofluorescence and chromatin immunoprecipitation were: affinity purified rabbit polyclonal antibodies against HIF-1a (Lyberopoulou et al, 2007), lipin 1 and lipin 2 (Grimsey et al, 2008), rabbit polyclonal antibodies against GFP (Mylonis et al, 2008) or anti-HIF-2a (Abcam, Cambridge, UK) and mouse monoclonal antibodies against actin or tubulin (Millipore, Billerica, MA, USA). Analysis of total cellular proteins by immunoblotting and immunofluorescence microscopy were carried out as HIF-1 regulates lipin 1 3491 previously described (Grimsey et al, 2008;Lyberopoulou et al, 2007;Mylonis et al, 2008;Mylonis et al, 2006).…”

“…Analysis of total cellular proteins by immunoblotting and immunofluorescence microscopy were carried out as HIF-1 regulates lipin 1 3491 previously described (Grimsey et al, 2008;Lyberopoulou et al, 2007;Mylonis et al, 2008;Mylonis et al, 2006). To visualize lipid droplets after usual immunofluorescence microscopy procedure cells were stained with Nile Red (Sigma; 0.1 mg in PBS) for 15 minutes, washed with PBS and mounted on slides (Greenspan et al, 1985).…”

“…Following steps were performed as previously described (Braliou et al, 2008) using a polyclonal anti-HIF-1a antibody (Lyberopoulou et al, 2007) or rabbit IgG. The region 22916 to 22686 of the hLPIN1 promoter was amplified from immunoprecipitated chromatin or input samples using primers listed in supplementary material Table S1.…”

The role of E2F7 and E2F8 in squamous differentiation, UV- and chemotherapy-induced responses in keratinocytes

“…Curiously, RacGAP1 has been shown to interact physically with HIF1α and regulates its transcriptional activity (Lyberopoulou, Venieris et al 2007). …”