Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen.
HeLa cells contain a serine/threonine protein kinase (DNA-PK) that is strongly activated in vitro by low concentrations of double-stranded DNA (dsDNA). Activation was specific for dsDNA; both natural DNAs and synthetic oligonucleotides functioned as kinase activators. The fact that DNA-PK activity was rapidly inhibited by incubation with dsDNA and ATP suggests that DNA-PK activity also may be regulated by autophosphorylation. During gel filtration, DNA-PK activity behaved as a 350-kDa protein, and highly purified DNA-PK contained a dsDNA-binding, 350-kDa polypeptide that was phosphorylated in a dsDNA-dependent manner. We conclude that this 350-kDa polypeptide is likely to be DNA-PK. Previously we showed that the dsDNA-activated kinase phosphorylates two threonines at the N terminus of hsp90 alpha (S. P. Lees-Miller and C. W. Anderson, J. Biol. Chem. 264:17275-17280, 1989). Here we show that DNA-PK also phosphorylates the simian virus 40 large tumor antigen, the mouse tumor-suppressor protein p53, the human Ku autoantigen, and two unidentified HeLa DNA-associated polypeptides of 52 and 110 kDa. Identification of these and other newly identified DNA-binding substrates suggest that the dsDNA-activated kinase may regulate transcription, DNA replication, or cell growth.
Lees-Miller
S P
SP
Biology Department, Brookhaven National Laboratory, Upton, New York 11973.
Chen
Y R
YR
Anderson
C W
CW
D000595:Amino Acid Sequence; D000818:Animals; D034961:Antigens, Nuclear; D000952:Antigens, Polyomavirus Transforming; D000954:Antigens, Surface; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D000072200:Ku Autoantigen; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D034961:Antigens, Nuclear; D000952:Antigens, Polyomavirus Transforming; D000954:Antigens, Surface; D004268:DNA-Binding Proteins; D016159:Tumor Suppressor Protein p53; D011494:Protein Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen