DNA-PK;
Ku70;
Ku80;
staurosporine;
protein phosphorylation;
okadaic acid;
PP2A;
non-homologous end joining;
D O I:
10.1016/j.dnarep.2005.05.003
中图分类号:
Q3 [遗传学];
学科分类号:
071007 ;
090102 ;
摘要:
The Ku70/80 heterodimer is a major player in non-homologous end joining and the repair of DNA double-strand breaks. Studies suggest that once bound to a DNA double-strand break, Ku recruits the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) to form the DNA-dependent protein kinase holoenzyme complex (DNA-PK). We previously identified four DNA-PK phosphorylation sites on the Ku70/80 heterodimer: serine 6 of Ku70, serine 577 and 580 and threonine 715 of Ku80. This raised the interesting possibility that DNA-PK-dependent phosphorylation of Ku could provide a mechanism for the regulation of non-homologous end joining. Here, using mass spectrometry and phosphospecific antibodies we confirm that these sites are phosphorylated in vitro by purified DNA-PK. However, we show that neither DNA-PK nor the related protein kinase ataxia-telangiectasia mutated (ATM) is required for phosphorylation of Ku at these sites in vivo. Furthermore, Ku containing serine/threonine to alanine mutations at these sites was fully able to complement the radiation sensitivity of Ku negative mammalian cells indicating that phosphorylation at these sites is not required for non-homologous end joining. Interestingly, both Ku70 and Ku80 were phosphorylated in cells treated with the protein phosphatase inhibitor okadaic acid under conditions known to inactivate protein phosphatase 2A-like protein phosphatases. Moreover, okadaic acid-induced phosphorylation of Ku80 was inhibited by nanomolar concentrations of the protein kinase inhibitor staurosporine. These results suggest that the phosphorylation of Ku70 and Ku80 is regulated by a protein phosphatase 2A-like protein phosphatase and a staurosporine sensitive protein kinase in vivo, but that DNA-PK-mediated phosphorylation of Ku is not required for DNA double-strand break repair. (c) 2005 Elsevier B.V. All rights reserved.
机构:
WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105
ALESSI, DR
;
CUENDA, A
论文数: 0引用数: 0
h-index: 0
机构:
WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105
CUENDA, A
;
COHEN, P
论文数: 0引用数: 0
h-index: 0
机构:
WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105
COHEN, P
;
DUDLEY, DT
论文数: 0引用数: 0
h-index: 0
机构:
WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105
DUDLEY, DT
;
SALTIEL, AR
论文数: 0引用数: 0
h-index: 0
机构:
WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105
机构:
WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105
ALESSI, DR
;
CUENDA, A
论文数: 0引用数: 0
h-index: 0
机构:
WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105
CUENDA, A
;
COHEN, P
论文数: 0引用数: 0
h-index: 0
机构:
WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105
COHEN, P
;
DUDLEY, DT
论文数: 0引用数: 0
h-index: 0
机构:
WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105
DUDLEY, DT
;
SALTIEL, AR
论文数: 0引用数: 0
h-index: 0
机构:
WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105WARNER LAMBERT PARKE DAVIS,PARKE DAVIS PHARMACEUT RES DIV,DEPT SIGNAL TRANSDUCT,ANN ARBOR,MI 48105