title: Deformed epidermal autoregulatory factor-1 (DEAF1) interacts with the Ku70 subunit of the DNA-dependent protein kinase complex.
abstract: Deformed Epidermal Autoregulatory Factor 1 (DEAF1) is a transcription factor linked to suicide, cancer, autoimmune disorders and neural tube defects. To better understand the role of DEAF1 in protein interaction networks, a GST-DEAF1 fusion protein was used to isolate interacting proteins in mammalian cell lysates, and the XRCC6 (Ku70) and the XRCC5 (Ku80) subunits of DNA dependent protein kinase (DNA-PK) complex were identified by mass spectrometry, and the DNA-PK catalytic subunit was identified by immunoblotting. Interaction of DEAF1 with Ku70 and Ku80 was confirmed to occur within cells by co-immunoprecipitation of epitope-tagged proteins, and was mediated through interaction with the Ku70 subunit. Using in vitro GST-pulldowns, interaction between DEAF1 and the Ku70 subunit was mapped to the DEAF1 DNA binding domain and the C-terminal Bax-binding region of Ku70. In transfected cells, DEAF1 and Ku70 colocalized to the nucleus, but Ku70 could not relocalize a mutant cytoplasmic form of DEAF1 to the nucleus. Using an in vitro kinase assay, DEAF1 was phosphorylated by DNA-PK in a DNA-independent manner. Electrophoretic mobility shift assays showed that DEAF1 or Ku70/Ku80 did not interfere with the DNA binding of each other, but DNA containing DEAF1 binding sites inhibited the DEAF1-Ku70 interaction. The data demonstrates that DEAF1 can interact with the DNA-PK complex through interactions of its DNA binding domain with the carboxy-terminal region of Ku70 that contains the Bax binding domain, and that DEAF1 is a potential substrate for DNA-PK.
authors.affiliation: Department of Physiology, Southern Illinois University School of Medicine, Carbondale, Illinois, United States of America.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Jensik
authors.forename: Jodi I
authors.initials: JI
authors.lastname: Huggenvik
authors.forename: Michael W
authors.initials: MW
authors.lastname: Collard
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000882:Haplorhini; D006801:Humans; D000072200:Ku Autoantigen; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D014157:Transcription Factors
chemical_list: D034961:Antigens, Nuclear; C116013:DEAF1 protein, human; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D051747:DNA-Activated Protein Kinase; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen