title: DNA-PK contributes to the phosphorylation of AIRE: importance in transcriptional activity.
abstract: The autoimmune regulator (AIRE) protein is a key mediator of the central tolerance for tissue specific antigens and is involved in transcriptional control of many antigens in thymic medullary epithelial cells (mTEC). Mutations in the AIRE gene cause a rare disease named autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Here we report using GST pull-down assay, mass-spectrometry and co-immunoprecipitation that a heterotrimeric complex of DNA-Dependent Protein Kinase (DNA-PK), consisting of Ku70, Ku80 and DNA-PK catalytic subunit (DNA-PKcs), is a novel interaction partner for AIRE. In vitro phosphorylation assays show that the residues Thr68 and Ser156 are DNA-PK phosphorylation sites in AIRE. In addition, we demonstrate that DNA-PKcs is expressed in AIRE positive mTEC cell population and that introduction of mutations into the AIRE phosphorylation sites decrease the capacity of AIRE to activate transcription from reporter promoters. In conclusion, our results suggest that phosphorylation of the AIRE protein at Thr68 and Ser156 by DNA-PK influences AIRE transactivation ability and might have impact on other aspects of the functional regulation of the AIRE protein.
authors.affiliation: Molecular Pathology, University of Tartu, Tartu 50411, Estonia.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Liiv
authors.forename: Ana
authors.initials: A
authors.lastname: Rebane
authors.forename: Tõnis
authors.initials: T
authors.lastname: Org
authors.forename: Mario
authors.initials: M
authors.lastname: Saare
authors.forename: Julia
authors.initials: J
authors.lastname: Maslovskaja
authors.forename: Kai
authors.initials: K
authors.lastname: Kisand
authors.forename: Erkki
authors.initials: E
authors.lastname: Juronen
authors.forename: Leena
authors.initials: L
authors.lastname: Valmu
authors.forename: Matthew James
authors.initials: MJ
authors.lastname: Bottomley
authors.forename: Nisse
authors.initials: N
authors.lastname: Kalkkinen
authors.forename: Pärt
authors.initials: P
authors.lastname: Peterson
mesh_terms: D020816:Amino Acid Motifs; D034961:Antigens, Nuclear / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D000072200:Ku Autoantigen; D013058:Mass Spectrometry; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D011485:Protein Binding; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics*; D000096927:AIRE Protein
chemical_list: D034961:Antigens, Nuclear; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D000072200:Ku Autoantigen; D010768:Phosphoserine; D010769:Phosphothreonine; D014157:Transcription Factors; D000096927:AIRE Protein; C000606509:Xrcc6 protein, human