title: Identification of kinases phosphorylating 13 sites in the nuclear, DNA-binding protein NUCKS.
abstract: NUCKS is a vertebrate specific, nuclear and DNA-binding phospho protein. The protein is highly expressed in rapidly dividing cells, and is overexpressed in a number of cancer tissues. The phosphorylation of NUCKS is cell cycle and DNA-damage regulated, but little is known about the responsible kinases. By utilizing in vitro and in vivo phosphorylation assays using isolated NUCKS as well as synthetic NUCKS-derived peptides in combination with mass spectrometry, phosphopeptide mapping, phosphphoamino acid analyses, phosphospecific antibodies and the use of specific kinase inhibitors, we found that NUCKS is phosphorylated on 11 sites by CK2. At least 7 of the CK2 sites are phosphorylated in vivo. We also found that NUCKS is phosphorylated on two sites by ATM kinase and DNA-PK in vitro, and is phosphorylated in vivo by ATM kinase in γ-irradiated cells. All together, we identified three kinases phosphorylating 13 out of 39 in vivo phosphorylated sites in mammalian NUCKS. The identification of CK2 and PIKK kinases as kinases phosphorylating NUCKS in vivo provide further evidence for the involvement of NUCKS in cell cycle control and DNA repair.
authors.affiliation: University of Oslo, Institute of Basic Medical Sciences, Department of Biochemistry, P.O. Box 1112, Blindern N-0317, Oslo, Norway.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Grundt
authors.affiliation: University of Oslo, Department of Biosciences, P.O. Box 1066, Blindern N-0316, Oslo, Norway.
authors.forename: Bernd
authors.initials: B
authors.lastname: Thiede
authors.affiliation: University of Oslo, Institute of Basic Medical Sciences, Department of Biochemistry, P.O. Box 1112, Blindern N-0317, Oslo, Norway. Electronic address: a.c.ostvold@medisin.uio.no.
authors.forename: Anne Carine
authors.initials: AC
authors.lastname: Østvold
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D004249:DNA Damage / Q000502:physiology; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008322:Mammals / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology*; D011494:Protein Kinases / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011494:Protein Kinases; D064007:Ataxia Telangiectasia Mutated Proteins; D051747:DNA-Activated Protein Kinase
keywords: ATM kinase; CK2; Cell cycle; DNA repair; DNA-PK; Phosphorylation