Human Cyclin a Is Required for Mitosis until Mid Prophase

Abstract

We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not completely condense and daughter cells fuse. Remarkably, microinjecting cyclin A/CDK2 into S phase cells has no effect on progress through the following G2 phase or mitosis. In complementary experiments we have microinjected the amino terminus of p21Cip1/Waf1/Sdi1 (p21N) into cells to inhibit cyclin A/CDK2 activity. We find that p21N will prevent S phase or G2 phase cells from entering mitosis, and will cause early prophase cells to return to interphase. These results suggest that cyclin A/CDK2 is a rate-limiting component required for entry into mitosis, and for progress through mitosis until late prophase. They also suggest that cyclin A/CDK2 may be the target of the recently described prophase checkpoint.

Cyclin A/cdk2 coordinates centrosomal and nuclear mitotic events

“…A number of laboratories have provided evidence that G 2 phase cyclin A/cdk2 has a critical role in the timing of entry into mitosis (Furuno et al, 1999;Goldstone et al, 2001;Hu et al, 2001;Mitra and Enders, 2004;Fung et al, 2007;Gong et al, 2007), which is confirmed by our siRNA and cdk2-specific inhibitor studies. In agreement with these reports, the present study demonstrates that inhibition of G 2 phase cyclin A/cdk2 delays the cdc25-dependent activation (Mitra and Enders, 2004;Fung et al, 2007), and nuclear translocation (Gong et al, 2007) of cyclin B/cdk1.…”

“…Furthermore, cells from a Drosophila mutant lacking cyclin A arrest in G 2 after the maternal cyclin A has been exhausted (Knoblich and Lehner, 1993), indicating the importance of the cyclin A/cdk complex. Microinjection of active cyclin A/cdk2 into early G 2 phase HeLa cells promotes premature entry into mitosis, and it has been suggested that the complex is a rate-limiting component required for entry into and progression through mitosis until late prophase (Furuno et al, 1999).…”

“…Cyclin A/cdk2 appears to be involved in the activation (Furuno et al, 1999;Mitra and Enders, 2004), and possibly the stabilization of cyclin B, the latter by indirectly inhibiting the anaphase-promoting complex/cyclosome (APC/C) (Lukas et al, 1999). Cyclin A is itself destroyed by the APC/C at prometaphase (den Elzen and Pines, 2001) inferring that its activity is required until that time.…”

Cyclin B2 suppresses mitotic failure and DNA re-replication in human somatic cells knocked down for both cyclins B1 and B2

“…In mammalian cells, two A-type (A1 and A2) and two B-type (B1 and B2) cyclins are able to bind and activate CDK1. Cyclin A2/CDK complexes are necessary and sufficient to drive G 2 cells into prophase (Furuno et al, 1999) and are required for timely nuclear envelope breakdown (NEB) (Gong et al, 2007). However, the principal activator of CDK1 as a mitosis-promoting factor is cyclin B.…”

Identification of the Nuclear Localization Signal inXenopusCyclin E and Analysis of Its Role in Replication and Mitosis

“…In animal cells, several families of Cdks and cyclins have roles in cell cycle control. d-type cyclins complexed to Cdk4/Cdk6 regulate the decision to divide or differentiate, Cdk2/cyclin E and Cdk2/ cyclin A collaborate to initiate the events of S phase, and Cdk/cyclin A and Cdk1/cyclin B combine forces to trigger the wholesale reorganization of cellular components at mitosis (Girard et al, 1991;Pagano et al, 1992;Ohtsubo et al, 1995;Furuno et al, 1999;Hu et al, 2001;Nigg, 2001).Cdk/cyclin complexes are regulated by multiple mechanisms that ensure that they execute their functions at the correct time (Morgan, 1997); ubiquitylation and proteolysis of cyclins A and B are critical for M-phase exit (Wheatley et al, 1997;den Elzen and Pines, 2001; Geley et al, 2001), whereas defects in cyclin E proteolysis may be associated with certain cancers (Strohmaier et al, 2001). The subcellular localization of Cdk/cyclin complexes is also critical for faithful cell cycle control (Pines and Hunter, 1991;Hagting et al, 1998;Toyoshima et al, 1998;Pines, 1999;Alt et al, 2000;Draviam et al, 2001), and some progress has been made in identifying the mechanisms responsible, in particular those used by Cdk/cyclin complexes to shuttle between the cytoplasm and nucleus (Yang et al, 1998;Hagting et al, 1999;Moore et al, 1999;Takizawa et al, 1999).…”