Review
Control of mitosis by changes in the subcellular location of cyclin-B1–Cdk1 and Cdc25C

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Abstract

Nuclear events of mitosis are initiated when the protein kinase cyclin-B1–Cdk1 is translocated into the nucleus during prophase. Recent work has unveiled many of the mechanisms that govern the localization of cyclin-B1–Cdk1 and its regulator Cdc25C. Phosphorylation-dependent changes in the rate of nuclear import and export of these proteins help to control the onset of mitosis both in normal cells and in cells delayed before mitosis by DNA damage.

Introduction

Mitosis in vertebrate cells is thought to be triggered by the cyclin-dependent kinase Cdk1 (also known as Cdc2) [1], [2], [3], [4]. Cdk1 activation is a multi-step process that begins when it binds to its regulatory subunit cyclin B, whose levels rise during G2 and peak in mitosis. Before mitosis, cyclin-B–Cdk1 complexes are held in an inactive state by phosphorylation of Cdk1 at Thr14 and Tyr15, which is catalyzed by the protein kinases Wee1 (which phosphorylates Tyr15 only) and Myt1 (which phosphorylates both Thr14 and Tyr15). Dephosphorylation is carried out by the dual-specificity protein phosphatase Cdc25C.

Cdk1 activation at the onset of mitosis results from the concurrent inhibition of Wee1 and Myt1 and stimulation of the phosphatase Cdc25C. Activation of Cdc25C requires phosphorylation of several sites in the Cdc25C amino-terminal domain; this is catalyzed by at least two kinases. One of these kinases appears to be the Polo-related kinase (Plk): the Xenopus Plk homolog, Plx, phosphorylates and activates Cdc25C in vitro and is required for Cdc25C activation in frog egg extracts [5]. Activating phosphorylation of Cdc25C is also carried out by the Cyclin-B1–Cdk1 complex itself [6]. The ability of Cdk1 to activate its own activator provides the potential for a positive feedback loop in Cdk1 activation, whereby partial activation of Cdc25C (perhaps by Plk) could lead to complete and overwhelming Cdk1 activation. This positive feedback loop may be enhanced by the ability of Cdk1 to phosphorylate and inactivate Wee1 [7], [8]. There is also evidence that Cdk1 stimulates Plk activity in frog egg extracts, providing another potential mechanism for positive feedback [9].

There are two different cyclin B proteins in mammalian cells. Cyclin B2 is a non-essential protein that associates with the Golgi apparatus and probably plays a role in Golgi remodelling during mitosis [10], [11]. Cyclin B1 is an essential protein that is thought to be responsible for most of the other actions of Cdk1 in the cytoplasm and nucleus [11]. The localization of cyclin B1 is subject to extensive regulation and is the major focus of this review.

Section snippets

Control of cyclin B1 localization

During interphase, cyclin-B1–Cdk1 is found entirely in the cytoplasm, either in a soluble form or associated with the microtubule network and centrosomes [12], [13], [14]. In late prophase, most cyclin-B1–Cdk1 complexes are translocated rapidly from the cytoplasm to the nucleus, after which the nuclear envelope breaks down [15, [16]. A fraction of cyclin-B1–Cdk1 remains in the cytoplasm during prophase and is associated, at least in part, with the duplicated centrosomes as they separate [14].

Control of Cdc25C localization

Mitotic entry is also influenced by regulated changes in the location of the activating phosphatase Cdc25C. Although there remains some disagreement about the exact localization of Cdc25C during the cell cycle, most evidence now suggests that Cdc25C, like cyclin-B1–Cdk1, is localized in the cytoplasm during interphase and relocalizes to the nucleus during prophase [[29], [30], [31, [32, [33].

Cdc25C, like cyclin B1, shuttles continuously in and out of the nucleus, and its steady state

How regulated localization may influence Cdk1 activation

With a basic knowledge of cyclin B1 and Cdc25C localization in mind, we can now address some complex and unresolved questions about the biological importance of localization in the activation of Cdk1 and in the triggering of mitotic events.

What is the relationship between the nuclear translocation of cyclin-B1–Cdk1 and the process by which Cdk1 is abruptly activated at the onset of mitosis? One can imagine two alternatives: one, Cdk1 activation occurs in the cytoplasm and is completely

How regulated localization influences the entry into mitosis

Major mitotic events of the nucleus, such as nuclear envelope breakdown, are thought to be triggered by cyclin-B1–Cdk1-dependent phosphorylation of certain substrates in the nucleus (nuclear lamins, for example). Thus, it is not surprising that translocation of cyclin-B1–Cdk1 to the nucleus appears to be required for the stimulation of these events. The ability of cyclin B1 to stimulate nuclear envelope breakdown in frog oocytes is abolished by mutations in the CRS that prevent import of the

Inhibition of mitotic entry by DNA damage and incomplete DNA replication

In vertebrate cells, the presence of unreplicated DNA or DNA damage blocks the activation of cyclin-B1–Cdk1 complexes, thereby preventing the initiation of mitotic events until replication is complete or DNA damage is repaired (see review by Nancy Walworth on pp 697–704 of this issue). The inhibition of Cdk1 activation by these stimuli is due in part to the stabilization of Cdc25C phosphorylation at Ser216/287 [35], [43]. DNA damage and unreplicated DNA trigger signaling through pathways

Conclusions

In summary, recent work has shown that the translocation of cyclin B1 and Cdc25C into the nucleus probably plays an important role in governing the onset of nuclear mitotic events such as nuclear envelope breakdown; a role in cytoplasmic events, however, appears unlikely. Nuclear translocation of these proteins is governed by phosphorylation-dependent changes in their relative rates of nuclear export and import, but we know little about the kinases and phosphatases acting on these proteins and

Acknowledgements

The authors are very grateful to Bill Dunphy, Jim Ferrell, Sally Kornbluth, Jonathan Pines, Helen Piwnica-Worms and Mark Terasaki for many helpful insights and for sharing unpublished data.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • radical dot of special interest

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