DNA-dependent Protein Kinase Interacts with Antigen Receptor Response Element Binding Proteins NF90 and NF45

• Published in: The Journal of biological chemistry 1998-01, Vol.273 (4), p.2136-2145
• Main Authors: Ting, Nicholas S.Y., Kao, Peter N., Chan, Doug W., Lintott, Lauri G., Lees-Miller, Susan P.
• Format: Article
• Language: English
• Subjects: Antigens, Nuclear; Autoantigens - metabolism; DNA - metabolism; DNA Helicases; DNA-Activated Protein Kinase; DNA-Binding Proteins - metabolism; Eukaryotic Initiation Factor-2 - metabolism; Humans; Interleukin-2 - genetics; Ku Autoantigen; Macromolecular Substances; NFATC Transcription Factors; Nuclear Factor 45 Protein; Nuclear Factor 90 Proteins; Nuclear Proteins - metabolism; Phosphoproteins - metabolism; Phosphorylation; Promoter Regions, Genetic; Protein-Serine-Threonine Kinases - metabolism; Transcription Factors - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.4.2136

The DNA-dependent protein kinase (DNA-PK) is composed of a large catalytic subunit of approximately 470 kDa (DNA-PKcs) and the DNA-binding protein, Ku. Absence of DNA-PK activity confers sensitivity to x-rays and defects in both DNA double-strand break repair and V(D)J recombination. However, the precise function of DNA-PK in DNA double-strand break repair is not known. Here we show, using electrophoretic mobility shift assays, that polypeptides in a fraction purified from human cells interact with DNA-PK and stabilize the formation of a complex containing DNA-PKcs-Ku and DNA. Five polypeptides in this fraction have been identified by amino-terminal sequence analysis and/or immunoblotting. These proteins are NF90 and NF45, which are the 90- and 45-kDa subunits of a protein known to bind specifically to the antigen receptor response element of the interleukin 2 promoter, and the α, β, and γ subunits of eukaryotic translation initiation factor eIF-2. We also show that NF90, NF45, and eIF-2β are substrates for DNA-PK in vitro. In addition, recombinant NF90 promotes formation of a complex between DNA-PKcs, Ku, and DNA, and antibodies to recombinant NF90 or recombinant NF45 immunoprecipitate DNA-PKcs in vitro. Together, our data suggest that NF90, in complex with NF45, interacts with DNA-PKcs and Ku on DNA and that NF90 and NF45 may be important for the function of DNA-PK.