Microtubule dependency of p34cdc2 inactivation and mitotic exit in mammalian cells.
The protein kinase inhibitor 2-aminopurine induces checkpoint override and mitotic exit in BHK cells which have been arrested in mitosis by inhibitors of microtubule function (Andreassen, P. R., and R. L. Margolis. 1991. J. Cell Sci. 100:299-310). Mitotic exit is monitored by loss of MPM-2 antigen, by the reformation of nuclei, and by the extinction of p34cdc2-dependent H1 kinase activity. 2-AP-induced inactivation of p34cdc2 and mitotic exit depend on the assembly state of microtubules. During mitotic arrest generated by the microtubule assembly inhibitor nocodazole, the rate of mitotic exit induced by 2-AP decreases proportionally with increasing nocodazole concentrations. At nocodazole concentrations of 0.12 microgram/ml or greater, 2-AP induces no apparent exit through 75 min of treatment. In contrast, 2-AP brings about a rapid exit (t1/2 = 20 min) from mitotic arrest by taxol, a drug which causes inappropriate overassembly of microtubules. In control mitotic cells, p34cdc2 localizes to kinetochores, centrosomes, and spindle microtubules. We find that efficient exit from mitosis occurs under conditions where p34cdc2 remains associated with centrosomal microtubules, suggesting it must be present on these microtubules in order to be inactivated. Mitotic slippage, the natural reentry of cells into G1 during prolonged mitotic block, is also microtubule dependent. At high nocodazole concentrations slippage is prevented and mitotic arrest approaches 100%. We conclude that essential components of the machinery for exit from mitosis are present on the mitotic spindle, and that normal mitotic exit thereby may be regulated by the microtubule assembly state.
Andreassen
P R
PR
Institut de Biologie Structurale, Grenoble, France.
Margolis
R L
RL
D015075:2-Aminopurine / Q000494:pharmacology*; D000818:Animals; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors*; D002460:Cell Line; D006224:Cricetinae; D005434:Flow Cytometry; D007668:Kidney; D007700:Kinetics; D008870:Microtubules / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure*; D008938:Mitosis / Q000187:drug effects / Q000502:physiology*; D015739:Nocodazole / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D006656:Protamine Kinase / Q000378:metabolism; D015398:Signal Transduction
D015075:2-Aminopurine; D006656:Protamine Kinase; D016203:CDC2 Protein Kinase; D017239:Paclitaxel; D015739:Nocodazole