DNA-dependent protein kinase interacts with antigen receptor response element binding proteins NF90 and NF45.
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 alpha, beta, and gamma subunits of eukaryotic translation initiation factor eIF-2. We also show that NF90, NF45, and eIF-2 beta 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.
Ting
N S
NS
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Kao
P N
PN
Chan
D W
DW
Lintott
L G
LG
Lees-Miller
S P
SP
D034961:Antigens, Nuclear; D001324:Autoantigens / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics; D000072200:Ku Autoantigen; D046911:Macromolecular Substances; D050778:NFATC Transcription Factors; D051903:Nuclear Factor 45 Protein; D050779:Nuclear Factor 90 Proteins; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D034961:Antigens, Nuclear; D001324:Autoantigens; D004268:DNA-Binding Proteins; D015852:Eukaryotic Initiation Factor-2; C494013:ILF2 protein, human; D007376:Interleukin-2; D046911:Macromolecular Substances; D050778:NFATC Transcription Factors; D051903:Nuclear Factor 45 Protein; D050779:Nuclear Factor 90 Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D014157:Transcription Factors; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen