A Bifunctional Regulatory Element in Human Somatic Wee1 Mediates Cyclin A/Cdk2 Binding and Crm1-Dependent Nuclear Export

Li, Changqing; Andrake, Mark D.; Dunbrack, Roland L.; Enders, Greg H.

doi:10.1128/mcb.01876-08

Cited by 38 publications

(53 citation statements)

References 61 publications

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“…S2). This response is reminiscent of feedback by Cdk1 to prevent its own inhibition by phosphorylating Wee1 and Cdc25C, and suggests similar feedback between Cdk2 and Wee1/Cdc25A, because Cdk2 phosphorylates Wee1 and Cdc25A (33,34). Cyclin E abundance normally increases in S phase-arrested cells, and we found that cyclin E accumulated to high levels in HU-treated Cdk2 +/+ and Cdk2 +/− cells (Fig.…”

Section: Cdk2 Inhibitory Phosphorylationmentioning

confidence: 53%

Essential role for Cdk2 inhibitory phosphorylation during replication stress revealed by a human Cdk2 knockin mutation

Hughes

Sidorova

Swanger

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Cyclin-dependent kinases (Cdks) coordinate cell division, and their activities are tightly controlled. Phosphorylation of threonine 14 (T14) and tyrosine 15 (Y15) inhibits Cdks and regulates their activities in numerous physiologic contexts. Although the roles of Cdk1 inhibitory phosphorylation during mitosis are well described, studies of Cdk2 inhibitory phosphorylation during S phrase have largely been indirect. To specifically study the functions of Cdk2 inhibitory phosphorylation, we used gene targeting to make an endogenous Cdk2 knockin allele in human cells, termed Cdk2AF, which prevents Cdk2 T14 and Y15 phosphorylation. Cdk2AF caused premature S-phase entry, rapid cyclin E degradation, abnormal DNA replication, and genome instability. Cdk2AF cells also exhibited strikingly abnormal responses to replication stress, accumulated irreparable DNA damage, and permanently exited the cell cycle after transient exposure to S-phase inhibitors. Our results reveal the specific and essential roles of Cdk2 inhibitory phosphorylation in the successful execution of the replication stress checkpoint response and in maintaining genome integrity.Wee1 | cyclin A C yclin-dependent kinases (Cdks) are regulated by positive and negative phosphorylations (1). T14/Y15 phosphorylation by the Wee1 and Myt1 kinases inhibits Cdks by preventing ATP binding, and these conserved phosphorylations are highly regulated during the cell cycle and by signaling pathways (1, 2). Wee1 and Myt1 are opposed by cell division cycle 25 (Cdc25) phosphatases, which, in mammals, comprise three related enzymes that dephosphorylate T14/Y15 to promote Cdk activity (2-4).Wee1 and Cdc25 functions in mammals are best understood in G 2 /M, where Cdk1 inhibitory phosphorylation is required for mitotic progression and DNA damage responses. Cdk1 regulates Wee1 and Cdc25C by phosphorylation, which results in feedback loops that produce switch-like mitotic transitions (5). DNA damage pathways also use Cdk1 T14/Y15 phosphorylation to prevent mitotic entry by inhibiting Cdk1 (6, 7).Cdk2 is activated by cyclin E and cyclin A; cyclin E-Cdk2 regulates G 1 progression and S-phase entry, whereas cyclin A-Cdk2 acts later in S phase and mitosis (8, 9). Cdk2 is also regulated by Wee1 and Cdc25A, but the consequences of Cdk2 inhibitory phosphorylation are less well understood than for Cdk1 (10-13). Studies of Wee1 and Cdc25A have implicated Cdk2 inhibition in DNA replication and S-phase DNA damage checkpoints (14-17). However, the contributions of Cdk1 vs. Cdk2 to these phenotypes have been difficult to disentangle because manipulations such as Wee1 inhibition concurrently impact multiple Cdks. Indeed, a recent mouse model unexpectedly found that Cdk2 T14/Y15 phosphorylation is not required for DNA damage-induced cell cycle arrest, but instead has roles in S-phase entry and centrosome duplication (18).Cyclin E-Cdk2 activity peaks at the G 1 -S transition, and its periodicity results from regulated transcription and cyclin E degradation by the SCF (Skp1/Cul1/F-box protein)-Fbw7...

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Section: Cdk2 Inhibitory Phosphorylationmentioning

confidence: 53%

Essential role for Cdk2 inhibitory phosphorylation during replication stress revealed by a human Cdk2 knockin mutation

Hughes

Sidorova

Swanger

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…2, A and B). Importantly, kinases implicated previously in Wee1 turnover, such as Cdk1-cyclin B1, CK2, PLK1, or Cdk2-cyclin A2, were not inhibited by SR-653234 (7,8,12,18). Dose response measurements of kinases that were inhibited by SR-653234 by 65% or more in single-dose measurements provided evidence that the IC 50 s for inhibiting LKB1, FLT3, CK1␦, and CK1⑀ were 92, 100, 161, and 540 nM, respectively (Fig.…”

Section: Sr-653234 Selectively Stabilizesmentioning

confidence: 94%

Casein Kinase 1δ-dependent Wee1 Protein Degradation

Penas

Ramachandran

Simanski

et al. 2014

Journal of Biological Chemistry

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Background:Wee1 is an essential gene in mammals that encodes the cell cycle and cancer associated protein Wee1 kinase. Results: Inhibition of CK1␦ kinase increases the levels of Wee1 protein kinase. Conclusion: Casein kinase 1␦ is required for Wee1 degradation in HeLa cells and mouse embryonic fibroblasts. Significance: This is a previously unappreciated role for CK1␦ in controlling Wee1 degradation and cell cycle progression.

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“…Alternatively, the S472A and L483F mutations may disrupt binding to another protein required for Wee1 turnover. Indeed, a recent paper demonstrates that cyclin A1/Cdk2 binds somatic Wee1 kinase at multiple sites including Leu 483 and Arg 611 (25). These two sites were identified via our mutagenesis approach as required for Wee1 turnover ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Activation Domain-dependent Degradation of Somatic Wee1 Kinase

Owens

Simanski

Squire

et al. 2010

Journal of Biological Chemistry

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Cell cycle progression is dependent upon coordinate regulation of kinase and proteolytic pathways. Inhibitors of cell cycle transitions are degraded to allow progression into the subsequent cell cycle phase. For example, the tyrosine kinase and Cdk1 inhibitor Wee1 is degraded during G 2 and mitosis to allow mitotic progression. Previous studies suggested that the N terminus of Wee1 directs Wee1 destruction. Using a chemical mutagenesis strategy, we report that multiple regions of Wee1 control its destruction. Most notably, we find that the activation domain of the Wee1 kinase is also required for its degradation. Mutations in this domain inhibit Wee1 degradation in somatic cell extracts and in cells without affecting the overall Wee1 structure or kinase activity. More broadly, these findings suggest that kinase activation domains may be previously unappreciated sites of recognition by the ubiquitin proteasome pathway.The tyrosine kinase Wee1 inhibits mitotic entry, and its degradation is essential for exit from the G 2 phase of the cell cycle (1). Here, Wee1 inactivates the mitosis-specific cyclin-dependent kinase Cdk1-cyclin B complex during the S and G 2 phases by phosphorylating Cdk1 at tyrosine 15. However, Wee1 activity is opposed by the phosphatase Cdc25, which removes phosphate from tyrosine 15 on Cdk1, thereby activating Cdk1-cyclin B at the G 2 -M transition (2). A major mechanism that tips the balance toward Cdc25 and mitotic entry is Wee1 degradation during G 2 , and as this occurs active Cdc25 and Cdk1-cyclin B form a positive feedback loop, which ensures that mitotic entry is unidirectional (3, 4). Thus, Wee1 degradation is an essential component of the Cdk1 activation circuit.Wee1 degradation has been observed in Saccharomyces cerevisiae, Xenopus egg extracts, and somatic cells (5-11). In Xenopus, the nuclei are required for proper Wee1 degradation, and the completion of DNA replication is required to achieve maximal rates of Wee1 degradation (6). Thus, Wee1 degradation is part of a sensing mechanism that signals the completion of the DNA replication phase, ensuring proper timing of entry into mitosis. For example, when DNA replication stalls, this fail-safe mechanism allows defects to be corrected before cells enter mitosis. Indeed, current theories suggest that many cancer cells have ineffective checkpoint pathways that cause them to divide with incompletely replicated DNA, which leads to genomic instability (12). Consistent with this notion are the findings that wee1 knock-out mice are not viable and that wee1 Ϫ/Ϫ cells undergo mitotic catastrophe (13).Wee1 phosphorylation is required for its degradation and is regulated by the DNA replication checkpoint (6, 10). Here in the presence of the DNA replication checkpoint, Wee1 phosphorylation is negligible, whereas when DNA replication proceeds normally, Wee1 phosphorylation and degradation and mitotic entry are normal.One aspect of Wee1 degradation conserved between embryonic and somatic cell cycles is the requirement for SCF 3 ubiquitin ligases th...

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A Bifunctional Regulatory Element in Human Somatic Wee1 Mediates Cyclin A/Cdk2 Binding and Crm1-Dependent Nuclear Export

Cited by 38 publications

References 61 publications

Essential role for Cdk2 inhibitory phosphorylation during replication stress revealed by a human Cdk2 knockin mutation

Essential role for Cdk2 inhibitory phosphorylation during replication stress revealed by a human Cdk2 knockin mutation

Casein Kinase 1δ-dependent Wee1 Protein Degradation

Activation Domain-dependent Degradation of Somatic Wee1 Kinase

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