Figure 3.

Aurora A activity is not sufficient to activate Plk1 during the DDR. (a) Cells were released from a single thymidine block as indicated and treated for 1 h with 10 μM etoposide at the indicated time. Aurora A and Plk1 were immunoprecipitated and samples were blotted for the indicated proteins. (b) UT2R cells stably expressing tetracycline-inducible FLAG-Aurora A-wt, -K162R or -T288E were treated over night with tetracycline where indicated and expression was anlyzed by western blotting. (c) FLAG-Aurora A-wt, -K162R or -T288E from tetracycline-inducible U2TR cells were immunoprecipitated from a population of mitotic cells (mitotic shake-off) or DNA damage arrested cells (DNA damage) and assayed for activity using recombinant H3 as a substrate and probed for phosphorylation at S10. Lanes were present on the same blot but pasted together for comparison as indicated by the dashed lines. (d) U2TR cells expressing tetracycline-inducible FLAG-Aurora A-T288E were treated as indicated and blotted for the indicated proteins. (e) The Plk1 FRET-based biosensor was transiently transfected in cells stably expressing tetracycline-inducible Plk1-wt, Plk1-T210D, Aurora A-wt or Aurora A-T288E. Expression was induced with tetracycline and individual cells were treated as in 1B.