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Characterization of a new SUMO-1 nuclear body (SNB) enriched in pCREB, CBP, c-Jun in neuron-like UR61 cells

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Abstract

The neuron-like UR61 cell is a stable PC12 subline that contains a mouse N-ras oncogene. Dexamethasone (Dex) treatment induces a neuron-like differentiation, which is associated with neuritogenesis and nuclear expression of the glucocorticoid receptor and c-Jun. In differentiated UR61 cells, small ubiquitin-like modifiers 1 (SUMO-1) is concentrated in a new category of SUMO-1 nuclear bodies (SNBs) distinct from promyelocytic leukemia (PML) bodies by their large size and absence of PML protein. SNBs are 1 to 3 μm in diameter and exhibit a fine granular texture by electron microscopy. They are free of splicing factors and transcription foci and show spatial associations with Cajal bodies. In addition to SUMO-1 and the E2-conjugating enzyme Ubc9, which is essential for sumoylation, SNBs concentrate the transcriptional regulators CBP, CREB, and c-Jun. Moreover, transfection experiments demonstrate that SNBs accumulate the active conjugating form of SUMO-1 but not the conjugation defective variant of SUMO-1, supporting that SNBs are sites of sumoylation. Our results suggest that SNBs play a role in the control of the nucleoplasmic concentration of transcription regulators involved in neuroprotection and survival of the UR61 cells.

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Acknowledgements

We are grateful to Professor Angus I Lamond (University of Dundee, Scotland) for providing the anti-coilin antibody and Dr. Joana M. Desterro (IMM of Lisbon, Portugal) for providing pGFP-SUMO-1 plasmid constructs. This study was supported by the “Direccion General de Investigacion Cientifica” (Spain; BFU2005-01030), the “Instituto de Salud Carlos III (Spain; CIBERNED), and “Fundación Marqués de Valdecilla” in Santander (Spain; API05/04).

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Authors and Affiliations

  1. Department of Anatomy and Cell Biology, Biomedicine Unit, CSIC, University of Cantabria, Santander, Spain

    Rocio Bengoechea, Olga Tapia, Miguel Lafarga & Maria T. Berciano

  2. Department of Molecular Biology, Biomedicine Unit, CSIC, University of Cantabria, Santander, Spain

    José P. Vaqué

  3. Division of Gene Regulation and Expression,Wellcome Trust Biocentre, University of Dundee, DD1 5EH, Dundee, UK

    Joaquín Navascués

  4. Department of Anatomy and Cell Biology, Faculty of Medicine, University of Cantabria, Avd. Cardenal Herrera Oria s/n, 39011, Santander, Spain

    Miguel Lafarga

Authors
  1. Joaquín Navascués
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  2. Rocio Bengoechea
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  3. Olga Tapia
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  4. José P. Vaqué
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  5. Miguel Lafarga
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  6. Maria T. Berciano
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Correspondence to Miguel Lafarga.

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Communicated by G. Matera

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Supplementary Fig. 1Supplementary Fig. 2

Dex treatment induces neuron-like differentiation of UR61 cells into a sympathetic neuron-like phenotype. Combination of propidium iodide cytochemical staining for nucleic acids and phalloidin-FITC labeling shows euchromatic nuclei with prominent nucleoli (a), and growing neurites after 36 hours of Dex treatment (b). (c and d) Double labeling for β-actin mRNA and SMN protein reveals the concentration of the β-actin mRNA in the cell nucleus, growing neurites and growth cones (c), whereas SMN is accumulated in a CB and throughout the cytoplasm and neurites with higher concentration at the growth cones (d). (e and g) Neuron-like differentiation of UR61 cells is also associated with nuclear expression of GR (e) and c-Jun (g). Neurites are counterstained with phalliodin-FIT (f and h). Scale bar =  5 μm(GIF 789 kb)

Immunoblot of UR61 cell lysates using the rabbit polyclonal anti-PML antibody H-238 (Santa Cruz, USA). Immunolabeling produces a single band of approximately 64 kD, as is illustrated in the technical specification sheet of the manufacturer. Preparation of UR61 cell lysates and immunoblotting were performed as descrisbed previously (Navascues et al., 2004)(GIF 400 kb)

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Navascués, J., Bengoechea, R., Tapia, O. et al. Characterization of a new SUMO-1 nuclear body (SNB) enriched in pCREB, CBP, c-Jun in neuron-like UR61 cells. Chromosoma 116, 441–451 (2007). https://doi.org/10.1007/s00412-007-0107-7

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