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 activit y 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) cell s infected with recombinant vaccinia viruses directing the synthesis o f human p70 and p80, Pulse-chase analysis and density gradient centrif ugation revealed a pool of free p70 subunits in K562 cells that dimeri zed within minutes with newly synthesized p80, whereas Ku became assoc iated with newly synthesized p350 1 to 4h after the onset of p70/p80 h eterodimer assembly. A stable pool of free p80 subunits was not detect ed, and newly synthesized p80 was degraded rapidly (t(1/2) < 1.5 h) un less 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 K u-deficient RK13 or L-929 cells infected with recombinant vaccinia vir uses 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 < 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 > 16 h in RK13 cell s, In contrast, the tv, of unassembled p70 was < 1.5 h in L-929 cells, whereas the t, of p70/p80 heterodimers was similar to that in RK13 ce lls, suggesting that the turnover of p70 was regulated differently in RK13 and K562 than in L-929 cells. Taken together with previous observ ations that the free p70 subunit undergoes nuclear transport and has s imilar 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 bind ing 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 implication s for the regulation of DNA-PK activity.