Phosphorylation of a 72-kDa nucleoprotein (NP-72) in HL-60 cells is mediated by the double-stranded DNA-dependent protein kinase (DNA-PK).
Nuclear extracts prepared from human leukemic cells grown in the presence of cell differentiation-inducing agents showed a significant increase in the phosphorylation of a nuclear protein referred to as NP-72. The NP-72 phosphorylation was greatly increased by the addition of purified HeLa DNA-PK and was reduced by the inclusion of a DNA-PK-specific monoclonal antibody to the assay mixture. Phosphoamino acid analysis showed serine (60%) and threonine (40%) residues to be the phosphate acceptors. Western blot analysis of control and TPA-treated nuclear extracts detected multiple immunoreactive protein bands. The two most prominent species migrated as 300- and 210-kD proteins on SDS-PAGE, possibly representing an intact and a processed form of DNA-PK. The 300-kD form of DNA-PK was only detected in TPA-treated nuclear extracts, raising the possibility that cell differentiation is associated with the down-regulation and/or the inhibition of a protease(s) capable of regulating DNA-PK turnover.
Konno-Sato
S
S
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla 10595.
Wu
J M
JM
Carter
T H
TH
D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute; D008970:Molecular Weight; D009687:Nuclear Proteins; D009698:Nucleoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000276:immunology / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009698:Nucleoproteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D013755:Tetradecanoylphorbol Acetate