Abstract
Cell cycle regulation, especially faithful DNA replication and mitosis, are crucial to maintain genome stability. Cyclin-dependent kinase (CDK)/cyclin complexes drive most processes in cellular proliferation. In response to DNA damage, cell cycle surveillance mechanisms enable normal cells to arrest and undergo repair processes. Perturbations in genomic stability can lead to tumor development and suggest that cell cycle regulators could be effective targets in anticancer therapy. However, many clinical trials ended in failure due to off-target effects of the inhibitors used. Here, we investigate in vivo the importance of WEE1- and MYT1-dependent inhibitory phosphorylation of mammalian CDK1. We generated Cdk1AF knockin mice, in which two inhibitory phosphorylation sites are replaced by the non-phosphorylatable amino acids T14A/Y15F. We uncovered that monoallelic expression of CDK1AF is early embryonic lethal in mice and induces S phase arrest accompanied by γH2AX and DNA damage checkpoint activation in mouse embryonic fibroblasts (MEFs). The chromosomal fragmentation in Cdk1AF MEFs does not rely on CDK2 and is partly caused by premature activation of MUS81-SLX4 structure-specific endonuclease complexes, as well as untimely onset of chromosome condensation followed by nuclear lamina disassembly. We provide evidence that tumor development in liver expressing CDK1AF is inhibited. Interestingly, the regulatory mechanisms that impede cell proliferation in CDK1AF expressing cells differ partially from the actions of the WEE1 inhibitor, MK-1775, with p53 expression determining the sensitivity of cells to the drug response. Thus, our work highlights the importance of improved therapeutic strategies for patients with various cancer types and may explain why some patients respond better to WEE1 inhibitors.
Highlights
Proliferation of eukaryotic cells requires faithful replication and segregation of the genetic material during cell cycle progression and mitosis
To what we previously observed for Cdk1 knockout adult mice [2], animals expressing CDK1AF died within 5–6 days after tamoxifen administration, indicating that CDK1AF expression is lethal in adult animals
These in vivo observations suggest that the timely control of CDK1 activity via its inhibitory phosphorylation on T14 and Y15 is essential during the embryogenesis and adult life to prevent the formation of DNA breaks and the onset of apoptosis
Summary
Proliferation of eukaryotic cells requires faithful replication and segregation of the genetic material during cell cycle progression and mitosis. CDK activity is temporally and spatially regulated through the cell cycle and requires binding to cyclin partners [3], as well as posttranslational modifications. WEE1dependent phosphorylation on the Y15 residue prevents unscheduled entry into mitosis by keeping CDK1/cyclin B1 complexes in a low activity state [10,11,12,13]. The dual-specificity phosphatase CDC25 regulates the mitotic onset by the abrupt dephosphorylation of T14 and Y15, triggering complete activation of CDK1/cyclin B1 complexes [15,16,17]. The dephosphorylation of CDK1 on its inhibitory residues is believed to be a rate-limiting step for its activation and entry into mitosis [18, 19]
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