Proteomic, cellular, and network analyses reveal new DUSP3 interactions with nucleolar proteins in HeLa cells.
DUSP3 (or Vaccinia virus phosphatase VH1-related; VHR) is a small dual-specificity phosphatase known to dephosphorylate c-Jun N-terminal kinases and extracellular signal-regulated kinases. In human cervical cancer cells, DUSP3 is overexpressed, localizes preferentially to the nucleus, and plays a key role in cellular proliferation and senescence triggering. Other DUSP3 functions are still unknown, as illustrated by recent and unpublished results from our group showing that this enzyme mediates DNA damage response or repair processes. In this study, we sought to identify new interactions between DUSP3 and proteins directly or indirectly involved in or correlated with its biological roles in HeLa cells exposed to gamma or UV radiation. By using GST-DUSP as bait, we pulled down interacting proteins and identified them by LC-MS/MS. Of the 46 proteins obtained, six hits were extensively validated by immune techniques; the proteins Nucleophosmin, HnRNP C1/C2, and Nucleolin were the most promising targets found to directly interact with DUSP3. We then analyzed the DUSP3 interactomes using physical protein-protein interaction networks using our hits as the seed list. The validated hits as well as unvalidated hits fluctuated on the DUSP3 interactomes of HeLa cells, independent of the time post radiation, which confirmed our proteomic and experimental data and clearly showed the proximity of DUSP3 to proteins involved in processes intimately related to DNA repair and senescence, such as Ku70 and Tert, via interactions with nucleolar proteins, which were identified in this study, that regulate DNA/RNA structure and functions.
Centro de Ciências Naturais e Humanas, Universidade Federal do ABC , Rua Santa Adélia, 166, Bairro Bangu, Santo Andre-SP 09210-170, Brazil.
Karine
K
Panico
Fabio Luis
FL
Forti
D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism; D002853:Chromatography, Liquid; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D054640:Dual Specificity Phosphatase 3 / Q000235:genetics / Q000378:metabolism*; D005720:Gamma Rays; D005786:Gene Expression Regulation; D006367:HeLa Cells; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D000090243:Nucleophosmin; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D019098:Telomerase / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays; D000097796:Nucleolin
D034961:Antigens, Nuclear; D004247:DNA; D004268:DNA-Binding Proteins; D054640:Dual Specificity Phosphatase 3; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D000072200:Ku Autoantigen; D009687:Nuclear Proteins; D000090243:Nucleophosmin; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D019098:Telomerase; D000097796:Nucleolin; C000716967:NPM1 protein, human; C509186:TERT protein, human; C517025:DUSP3 protein, human; C000606509:Xrcc6 protein, human