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First published online 8 May 2007
doi: 10.1242/jcs.03454

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NRAGE associates with the anti-apoptotic factor Che-1 and regulates its degradation to induce cell death

¹ Department of Experimental Medicine, Via Vetoio, Coppito 2, University of L'Aquila, 67100 L'Aquila, Italy
² Istituto di Neurobiologia e Medicina Molecolare, CNR, c/o EBRI, Via del Fosso di Fiorano 64, 00143 Rome, Italy
³ Istituto di Biologia e Patologia Molecolari, CNR, c/o Regina Elena Cancer Institute, Via delle Messi d'Oro 156, 00158 Rome, Italy
⁴ Laboratory "B", Regina Elena Cancer Institute, Via delle Messi d'Oro 156, 00158 Rome, Italy
⁵ AIRC-Roman Oncogenomic Center (ROC), Via delle Messi d'Oro 156, 00158 Rome, Italy

View larger version (31K): [in this window] [in a new window]   Fig. 1. NRAGE interacts with Che-1. (A) Schematic representation of Che-1 C-terminal deletion mutants and full-length proteins. Construct names are indicated on the left. (B) Yeast two-hybrid assay. AH109 yeast cells were co-transformed with the indicated constructs and plated onto SD media lacking leucine and tryptophan (–LW) to verify the expression of both bait (W+) and prey (L+) plasmids, or onto media lacking leucine, tryptophan, histidine and adenine (–LWHA) to examine the interaction between bait and prey proteins. (C) Western blot analysis of lysates from NIH3T3 cells, transfected with expression constructs for Myc-NRAGE or vector expressing Myc-tag only, immunoprecipitated (IP) with anti-Myc monoclonal antibody. Anti-Che-1 polyclonal antibody was used to co-immunoprecipitate endogenous Che-1 protein in Myc-NRAGE expressing cells. (D) CGNs obtained from day 8 postnatal (P8) rats cultured in vitro for 6 days were processed for co-immunoprecipitation assay. Immunoblotting with anti-NRAGE polyclonal antibody showed endogenous NRAGE protein in samples immunoprecipitated with anti-Che-1 rabbit serum (I.P.-Che-1). No immunoreactivity from samples immunoprecipitated with normal rabbit serum (I.P.-control) was detected. (E) Dual-label fluorescence microscopy was performed in fixed CGNs to obtain immunolocalization of endogenous NRAGE (green) and Che-1 (red), as described in Material and Methods. Extensive colocalization (yellow) between NRAGE and Che-1 is visualised by the merged-colour image.

View larger version (45K): [in this window] [in a new window]   Fig. 2. The NRAGE MHD domain is required for Che-1 binding. (A) Schematic representations of both Flag-tagged NRAGE full-length and its derived deletion mutants. Construct names and binding activities are indicated on the left. (B) Cell lysates from NIH3T3 cells co-transfected with expression vectors encoding the indicated Flag-NRAGE proteins and Myc-Che-1 were immunoprecipitated with anti-Flag antibody. Immunoprecipitated samples were immunoblotted with anti-Myc monoclonal antibody to identify NRAGE domains involved in Che-1 interaction (top) and with anti-Flag antibody for the presence of Flag-NRAGE deletion mutants. Note that, the Flag-NRAGE-g mutant molecule was not revealed in the blot shown here, but under different – more appropriate – gel condition (data not shown).

View larger version (59K): [in this window] [in a new window]   Fig. 3. NRAGE affects Che-1 cellular localization. For co-expression experiments, NIH3T3 cells were doubly transfected with EGFP-NRAGE and Myc-Che-1 or one of the two Myc-Che-1 deletion mutants (Myc-Che-1-C, Myc-Che-1-C). After 48 hours, cells were fixed and immuno-stained with antibody against Myc-epitopes as described in Materials and Methods. Sub-cellular distribution and colocalization of Myc-tagged proteins (red) with EGFP-NRAGE (green) were analysed using fluorescence microscopy. No overlapping staining was observed in cells co-expressing Myc-Che-1-C and EGFP-NRAGE fusion proteins. Note that, EGFP-NRAGE expression affects the nuclear localization of Myc-Che-1.

View larger version (43K): [in this window] [in a new window]   Fig. 4. NRAGE induces ubiquitin-dependent Che-1 degradation. (A) Three representative sections showing endogenous Che-1 in SY5Y cells transfected with expression vector expressing EGFP-NRAGE fusion protein, subjected to indirect immunofluorescence analysis as described in Materials and Methods. In the merged image, no fluorescence signal derived from Che-1 (red) was detectable in cells overexpressing EGFP-NRAGE (green). (B) Western blot analysis of HeLa cells transfected with either Myc-NRAGE or with empty control vector, harvested at the indicated times and analysed on SDS-PAGE. The immunoreactivity of Che-1 and Myc-NRAGE proteins was detected by probing blots with anti-Che-1 and anti-Myc antibodies, respectively. Samples were normalised for protein content by reprobing blots with anti -tubulin. (C) In vivo ubiquitylation assay was performed with HeLa cells transiently co-transfected with plasmids expressing HA-tagged ubiquitin and Myc-NRAGE. After transfection, cells were treated without or with 20 µM MG132 for 4 hours and subjected to immunoprecipitation (IP) and western blotting (WB) analysis with the indicated antibodies. Che-1-ubiquitin conjugates were detected by using anti-HA monoclonal antibody.

View larger version (31K): [in this window] [in a new window]   Fig. 5. Che-1 counteracts NRAGE induced apoptosis. (A) P19 cells were transfected with siRNA targeting Che-1 (iChe-1) or with a non-specific siRNA control pool (Smart). After 24 hours, apoptosis was induced with 1 µM retinoic acid (RA). Cell-death was measured by Trypan Blue exclusion after 48 hours. Transfected cells were subjected to western blot analysis to verify reduced Che-1 expression levels after siRNA treatment (top). Note that cells with low Che-1 levels are more responsive to RA treatment. (B) P19 cells were transfected with Myc-tag expression vectors for NRAGE, Che-1 and Che-1-C proteins. After 48 hours, cell death was measured by Trypan Blue exclusion. Each point represents the mean of three independent experiments performed in duplicate. Note that, in co-expressing cells Che-1 but not its deletion mutant Che-1-C abrogates the ability of NRAGE to induce cell death. (C) Whole-cell extracts from transfected P19 cells were subjected to western blot analysis. Immunoblotting with anti-Myc monoclonal antibody was performed to visualise expression of Myc-tagged proteins, NRAGE (upper band), Che-1 and Che-1-C (middle and lower bands, respectively). The same samples were monitored for endogenous level of activated caspase-3 using antibody against cleaved caspase-3. Samples were normalised for protein content by reprobing blots with a monoclonal antibody to -tubulin. (D) Caspase-3 activity was measured in P19 cells in which Myc-NRAGE was co-transfected with increasing amounts of Myc-Che-1, as indicated. Caspase activity is shown as the n-fold increase in caspase activation by comparing the levels of luciferase activity of transfected cells with those of untreated control cells. Equal numbers of cells were analysed. Each point represents the mean of three different experiments performed in triplicate.

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