Role of free p70 (Ku) subunit in posttranslational stabilization of newly synthesized p80 during DNA-dependent protein kinase assembly.

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 (t1/2 < 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 t1/2 of the free recombinant human p80 subunit expressed in RK13 cells was < 1.5 h unless it was "rescued" by dimerization with p70. The t1/2 of human p70 as well as p70/p80 heterodimers was > 16 h in RK13 cells.(ABSTRACT TRUNCATED AT 250 WORDS)