Role of free p70 (Ku) subunit in posttranslational stabilization of newly synthesized p80 during DNA-dependent protein kinase assembly
Satoh, M.; Wang, J.; Reeves, W.H.
European Journal of Cell Biology 66(2): 127-135
1995
ISSN/ISBN: 0171-9335 PMID: 7774599 Document Number: 444276
The Ku antigen (p70/p80 heterodimer) is the DNA binding component of a DNA-dependent serine/threonine kinase (DNA-PK), the catalytic activity of which is carried by a 350 kDa polypeptide (p350). In the present studies, the assembly of p70, p80, and p350 was investigated in human K562 (erythroleukemia) cells, and rabbit (RK13) or murine (L-929) cells infected with recombinant vaccinia viruses directing the synthesis of human p70 and p80. Pulse-chase analysis and density gradient centrifugation revealed a pool of free p70 subunits in K562 cells that dimerized within minutes with newly synthesized p80, whereas Ku became associated with newly synthesized p350 1 to 4 h after the onset of p70/p80 heterodimer assembly. A stable pool of free p80 subunits was not detected, and newly synthesized p80 was degraded rapidly (t-1/2 lt 1.5 h) unless it became incorporated into a p70/p80 dimer. The explanation for the absence of unassembled p80 subunits in K562 cells was investigated further by expressing human p70 and p80 individually or together in Ku-deficient RK13 or L-929 cells infected with recombinant vaccinia viruses p70-vacc and/or p80- vacc. As in uninfected K562 cells, the t-1/2 of the free recombinant human p80 subunit expressed in RK13 cells was lt 1.5 h unless it was "rescued" by dimerization with p70. The t-1/2 of human p70 as well as p70/p80 heterodimers was gt 16 h in RK13 cells. In contrast, the t-1/2 of unassembled p70 was lt 1.5 h in L-929 cells, whereas the t-1/2 of p70/p80 heterodimers was similar to that in RK13 cells, suggesting that the turnover of p70 was regulated differently in RK13 and K562 than in L-929 cells. Taken together with previous observations that the free p70 subunit undergoes nuclear transport and has similar DNA binding properties to those of the p70/p80 heterodimer, the present studies raise the possibility that newly synthesized p70 may first become associated with chromatin followed by the sequential binding of p80 and p350. In view of the importance of the p70/p80 dimer in delivering p350 to its chromatin-bound substrates, the differences in Ku assembly and turnover in different cell types may have implications for the regulation of DNA-PK activity.