A DNA-activated protein kinase from HeLa cell nuclei.
A DNA-activated protein kinase (DNA-PK) was purified from nuclei of HeLa cells. Activity was associated with a single high-molecular-mass (approximately-300,000 Da) polypeptide when analyzed by gel filtration, denaturing polyacrylamide gel electrophoresis, and Western immunoblotting using a monoclonal antibody that also inhibits enzyme activity. Nuclear localization was indicated by subcellular fractionation and confirmed by immunofluorescence on whole cells. Double-stranded DNA stimulated phosphorylation of the 300-kDa polypeptide in purified preparations as well as phosphorylation of the exogenous substrates alpha-casein, simian virus 40 large T antigen, and the human heat shock protein hsp90. Autophosphorylation led to inactivation of the enzyme. The phosphorylation of casein was stimulated over 30-fold by DNA and was specific for serine and threonine residues. Bovine serum albumin and histone H1 were poor substrates for DNA-PK, and no phosphorylation of immunoglobulin G or histones other than H1 was observed. Supercoiled or heat-denatured DNA and synthetic double-stranded RNA or RNA-DNA copolymers did not stimulate casein phosphorylation by DNA-PK. Interaction of the enzyme with DNA in the absence of exogenous substrates was demonstrated by thermal inactivation and gel mobility shifts. These characteristics identify DNA-PK as distinct from other protein kinases described in the literature and suggest that activation by DNA is an important feature of the enzyme's in vivo function.
Carter
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T
Department of Biological Sciences, St. John's University, Jamaica, New York 11439.
Vancurová
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I
Sun
I
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Lou
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W
DeLeon
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S
D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002467:Cell Nucleus / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007074:Immunoglobulin G; D007700:Kinetics; D008970:Molecular Weight; D011089:Polydeoxyribonucleotides / Q000494:pharmacology; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011089:Polydeoxyribonucleotides; D004247:DNA; D011494:Protein Kinases