Molecular properties, substrate specificity and regulation of DNA-dependent protein kinase from Raji Burkitt's lymphoma cells.
A double-stranded DNA-dependent protein serine/threonine kinase (DNA-PK) was purified from a nuclear extract of Raji Burkitt's lymphoma cells by a three-step column-chromatographic procedure. The main silver-stained band visualized after SDS/PAGE corresponded to an autophosphorylated polypeptide of about 350-kDa that represents the catalytic component. The existence of Ku DNA-binding protein as a regulatory component in the purified enzyme was revealed by Western blot/enzyme immunoassay and direct inhibition test with anti-Ku sera from the autoimmune patients. The DNA-PK catalyzed phosphorylation of synthetic peptides corresponding to Myc and RB proteins in a DNA-dependent manner, indicating that DNA-PK may recognize a second core-sequence motif Pro-Ser/Thr- in addition to the putative consensus sequences of -Ser/Thr-Gln. The level of enzyme activity was significantly higher in DMSO-induced G0/G1-arrested Raji cells as well as in the cells after release from DMSO than in the log-phase cells.
Watanabe
F
F
Department of Pathological Biochemistry, Tokyo Medical and Dental University, Japan.
Teraoka
H
H
Iijima
S
S
Mimori
T
T
Tsukada
K
K
D000595:Amino Acid Sequence; D002051:Burkitt Lymphoma / Q000201:enzymology*; D002460:Cell Line; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation; D006801:Humans; D007399:Interphase; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010455:Peptides; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases