[show abstract][hide abstract] ABSTRACT: Aneuploidy, an abnormal chromosome set, can ensue from failure of the spindle checkpoint, the safeguard mechanism that halts anaphase onset until mitotic spindle assembly. Inefficiency of cells to maintain the normal chromosome set across cell generations has been linked to tumorigenesis and senescence. Here we show that oxidative stress overrides the spindle checkpoint mechanism. Oxidant challenge of checkpoint-arrested cells led to proteolysis of the anaphase inhibitor securin and mitotic cyclins. This appeared consequent to loss of cyclin B-cdk1 activity caused by oxidant-induced reversal of cdk1 inhibitory phosphorylation. These observations may provide a link between aneuploidy occurrence and oxidative stress.
[show abstract][hide abstract] ABSTRACT: M-phase Promoting Factor (MPF; the cyclin B-cdk 1 complex) is activated at M-phase onset by removal of inhibitory phosphorylation of cdk1 at thr-14 and tyr-15. At M-phase exit, MPF is destroyed by ubiquitin-dependent cyclin proteolysis. Thus, control of MPF activity via inhibitory phosphorylation is believed to be particularly crucial in regulating transition into, rather than out of, M-phase. Using the in vitro cell cycle system derived form Xenopus eggs, here we show, however, that inhibitory phosphorylation of cdk1 contributes to control MPF activity during M-phase exit. By sampling extracts at very short intervals during both meiotic and mitotic exit, we found that cyclin B1-associated cdk1 underwent transient inhibitory phosphorylation at tyr-15 and that cyclin B1-cdk1 activity fell more rapidly than the cyclin B1 content. Inhibitory phosphorylation of MPF correlated with phosphorylation changes of cdc25C, the MPF phosphatase, and physical interaction of cdk1 with wee1, the MPF kinase, during M-phase exit. MPF down-regulation required Ca(++)/calmodulin-dependent kinase II (CaMKII) and cAMP-dependent protein kinase (PKA) activities at meiosis and mitosis exit, respectively. Treatment of M-phase extracts with a mutant cyclin B1-cdk1AF complex, refractory to inhibition by phosphorylation, impaired binding of the Anaphase Promoting Complex/Cyclosome (APC/C) to its co-activator Cdc20 and altered M-phase exit. Thus, timely M-phase exit requires a tight coupling of proteolysis-dependent and proteolysis-independent mechanisms of MPF inactivation.
[show abstract][hide abstract] ABSTRACT: The spindle checkpoint prevents anaphase onset until completion of mitotic spindle assembly by restraining activation of the ubiquitin ligase anaphase-promoting complex/cyclosome-Cdc20 (APC/CCdc20). We show that the spindle checkpoint requires mitotic cyclin-dependent kinase (cdk) activity. Inhibiting cdk activity overrides checkpoint-dependent arrest in Xenopus egg extracts and human cells. Following inhibition, the interaction between APC/C and Cdc20 transiently increases while the inhibitory checkpoint protein Mad2 dissociates from Cdc20. Cdk inhibition also overcomes Mad2-induced mitotic arrest. In addition, in vitro cdk1-phosphorylated Cdc20 interacts with Mad2 rather than APC/ C. Thus, cdk activity is required to restrain APC/CCdc20 activation until completion of spindle assembly.
Genes & Development 11/2003; 17(20):2520-5. · 12.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mitosis requires cyclin-dependent kinase (cdk) 1-cyclin B activity . Exit from mitosis depends on the inactivation of the complex by the degradation of cyclin B . Cdk2 is also active during mitosis [3, 4]. In Xenopus egg extracts, cdk2 is primarily in complex with cyclin E, which is stable . At the end of mitosis, downregulation of cdk2-cyclin E activity is accompanied by inhibitory phosphorylation of cdk2 . Here, we show that cdk2-cyclin E activity maintains cdk1-cyclin B during mitosis. At mitosis exit, cdk2 is inactivated prior to cdk1. The loss of cdk2 activity follows and depends upon an increase in protein kinase A (PKA) activity. Prematurely inactivating cdk2 advances the time of cyclin B degradation and cdk1 inactivation. Blocking PKA, instead, stabilizes cdk2 activity and inhibits cyclin B degradation and cdk1 inactivation. The stabilization of cdk1-cyclin B is also induced by a mutant cdk2-cyclin E complex that is resistant to inhibitory phosphorylation. P21-Cip1, which inhibits both wild-type and mutant cdk2-cyclin E, reverses mitotic arrest under either condition. Our findings indicate that the proteolysis-independent downregulation of cdk2 activity at the end of mitosis depends on PKA and is required to activate the proteolysis cascade that leads to mitosis exit.
Current Biology 09/2001; 11(15):1221-6. · 9.49 Impact Factor