Cdk2 Kinase Is Required for Entry into Mitosis as a Positive Regulator of Cdc2–Cyclin B Kinase Activity

Abstract

In higher eukaryotes, Cdk2 kinase plays an essential role in regulating the G1-S transition. Here, we use cycling Xenopus egg extracts to examine the requirement of Cdk2 kinase on progression into mitosis. Interestingly, when Cdk2 kinase activity is inhibited by the Cdk-specific inhibitor, p21Cip1, a block to mitosis occurs, and inactive Cdc2-cyclin B accumulates. This block occurs in the absence of nuclei and is not due to direct inhibition of Cdc2 by Cip. Importantly, this block to mitosis is reversible by restoring Cdk2-cyclin E kinase activity to a Cip-treated cycling extract. Moreover, immunodepletion of Cdk2 from interphase extracts prevents activation of Cdc2 upon the addition of exogenous cyclin B. Thus, our data show that Cdk2 kinase is a positive regulator of Cdc2-cyclin B complexes and establish a link between Cdk2 kinase and cell cycle progression into mitosis.

p21Cip1/WAF1 is important for differentiation and survival of U937 cells

“…Furthermore, our analysis showed upregulation of Cdc2 activity 24 h after antisense-p21 induction, which was followed by apoptosis within 48 h. The expression level of Cdc2 did not change during apoptosis by antisense-p21 induction, thus ruling out a possible involvement of de novo induction of Cdc2 protein in the upregulation of the activity of this kinase. Since the kinase activity of Cdc2-Cyclin B is positively regulated by Cdk2 kinase, 21 Cdc2 must have been activated through the release of Cdk2 activity caused by downregulation of p21. While further studies are necessary in order to elucidate the mechanism of Cdc2 activation, it is conceivable that inappropriate activation of Cdc2 kinase might cause apoptosis of differentiated U937 cells.…”

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

“…When cyclin E levels are reduced by immunodepletion of egg extracts, the resulting preparations require higher levels of cyclin B than normal to promote nuclear envelope breakdown and chromosome condensation (Guadagno and Newport, 1996). We wondered whether the subcellular location of cyclin E was important for this effect.…”

“…Five reports using such methods have defined multiple roles of Cdk2/cyclin E in egg extracts. Cdk2/cyclin E is capable of providing all the Cdk activity necessary to support a single round of chromosomal DNA replication (Jackson et al, 1995;Strausfeld et al, 1996), plays a role in centrosome duplication (Hinchcliffe et al, 1999;Lacey et al, 1999), and is important for maintaining conditions permissive for cyclin B to activate Cdk1 and bring about mitosis (Guadagno and Newport, 1996). There are limits to the abilities of Cdk2/cyclin E, however, for it is unable to trigger entry into mitosis in the absence of other Cdk/cyclin complexes (Strausfeld et al, 1996), although a recent article claimed that it influenced the duration of M phase (D'Angiolella et al, 2001).…”

“…We also provide evidence that Cdk2/cyclin E must accumulate to high concentrations in the nucleus to trigger DNA replication. Nuclear accumulation, however, is not necessary for Cdk2/cyclin E to perform all its functions, as revealed by the similar threshold level of cyclin B required to trigger entry into mitosis when wild-type cyclin E is replaced by Cdk2/cyclin E⌬NLS.The Role of Cdk2/Cyclin E at the Interphase/Mitosis Transition Guadagno and Newport (1996) showed that either addition of the p21 Cdk inhibitor or depletion of cyclin E impaired the ability of interphase Xenopus egg extracts to enter mitosis in response to added recombinant cyclin B. These results were interpreted to indicate that Cdk2/cyclin E, in addition to its roles during S phase, also operates at the interphase/mitosis transition in Xenopus eggs.…”

“…In this regard, it is interesting that structures to which Cdk2/cyclin E has been reported to localize, namely, the nucleus (Hua et al, 1997) and centrosomes (Hinchcliffe et al, 1999), are required for the normal kinetics of activation of Cdk1/cyclin B in fertilized Xenopus eggs (Perez-Mongiovi et al, 2000). Currently, it is unclear what substrates Cdk2/ cyclin E must phosphorylate to promote entry into mitosis transition, but as discussed by Guadagno and Newport (1996), Cdc25C would be an obvious candidate, for it is known that Cdk2/cyclin E can phosphorylate sites in the regulatory domain of Cdc25C leading to activation of its phosphatase activity (Izumi and Maller, 1995). In mammalian somatic cells, Cdk2/cyclin A is thought to regulate the timing of activation of Cdk1/cyclin B (Pagano et al, 1992;Furuno et al, 1999), but unlike the situation in Xenopus extracts, there is no evidence that Cdk2/cyclin E can perform a similar function.…”