As key regulators of the cell cycle, the cyclin-dependent kinases must be tightly regulated by extra- and intracellular signals. The activity of cyclin-dependent kinases is controlled by four highly conserved biochemical mechanisms, forming a web of regulatory pathways unmatched in its elegance and intricacy.

Principles of CDK regulation

Cisplatin, carboplatin and oxaliplatin are platinum-based drugs that are widely used in cancer chemotherapy. Platinum–DNA adducts, which are formed following uptake of the drug into the nucleus of cells, activate several cellular processes that mediate the cytotoxicity of these platinum drugs. This review focuses on recently discovered cellular pathways that are activated in response to cisplatin, including those involved in regulating drug uptake, the signalling of DNA damage, cell-cycle checkpoints and arrest, DNA repair and cell death. Such knowledge of the cellular processing of cisplatin adducts with DNA provides valuable clues for the rational design of more efficient platinum-based drugs as well as the development of new therapeutic strategies.

Cellular processing of platinum anticancer drugs.

The inability to repair DNA damage properly in mammals leads to various disorders and enhanced rates of tumour development. Organisms respond to chromosomal insults by activating a complex damage response pathway. This pathway regulates known responses such as cell-cycle arrest and apoptosis (programmed cell death), and has recently been shown to control additional processes including direct activation of DNA repair networks.

https://faculty.missouri.edu/~gatesk/DNADamageResponse.pdf

The DNA damage response: putting checkpoints in perspective

DNA damage is a relatively common event in the life of a cell and may lead to mutation, cancer, and cellular or organismic death. Damage to DNA induces several cellular responses that enable the cell either to eliminate or cope with the damage or to activate a programmed cell death process, presumably to eliminate cells with potentially catastrophic mutations. These DNA damage response reactions include: (a) removal of DNA damage and restoration of the continuity of the DNA duplex; (b) activation of a DNA damage checkpoint, which arrests cell cycle progression so as to allow for repair and prevention of the transmission of damaged or incompletely replicated chromosomes; (c) transcriptional response, which causes changes in the transcription profile that may be beneficial to the cell; and (d) apoptosis, which eliminates heavily damaged or seriously deregulated cells. DNA repair mechanisms include direct repair, base excision repair, nucleotide excision repair, double-strand break repair, and cross-link repair. The DNA damage checkpoints employ damage sensor proteins, such as ATM, ATR, the Rad17-RFC complex, and the 9-1-1 complex, to detect DNA damage and to initiate signal transduction cascades that employ Chk1 and Chk2 Ser/Thr kinases and Cdc25 phosphatases. The signal transducers activate p53 and inactivate cyclin-dependent kinases to inhibit cell cycle progression from G1 to S (the G1/S checkpoint), DNA replication (the intra-S checkpoint), or G2 to mitosis (the G2/M checkpoint). In this review the molecular mechanisms of DNA repair and the DNA damage checkpoints in mammalian cells are analyzed.

Molecular Mechanisms of Mammalian DNA Repair and the DNA Damage Checkpoints

After DNA damage, many cells appear to enter a sustained arrest in the G 2 phase of the cell cycle. It is shown here that this arrest could be sustained only when p53 was present in the cell and capable of transcriptionally activating the cyclin-dependent kinase inhibitor p21. After disruption of either the p53 or the p21 gene, γ radiated cells progressed into mitosis and exhibited a G 2 DNA content only because of a failure of cytokinesis. Thus, p53 and p21 appear to be essential for maintaining the G 2 checkpoint in human cells.

https://science.sciencemag.org/content/sci/282/5393/1497.full.pdf

Requirement for p53 and p21 to Sustain G2 Arrest After DNA Damage

Human Cdc25C is a dual-specificity protein phosphatase that controls entry into mitosis by dephosphorylating the protein kinase Cdc2. Throughout interphase, but not in mitosis, Cdc25C was phosphorylated on serine-216 and bound to members of the highly conserved and ubiquitously expressed family of 14-3-3 proteins. A mutation preventing phosphorylation of serine-216 abrogated 14-3-3 binding. Conditional overexpression of this mutant perturbed mitotic timing and allowed cells to escape the G2 checkpoint arrest induced by either unreplicated DNA or radiation-induced damage. Chk1, a fission yeast kinase involved in the DNA damage checkpoint response, phosphorylated Cdc25C in vitro on serine-216. These results indicate that serine-216 phosphorylation and 14-3-3 binding negatively regulate Cdc25C and identify Cdc25C as a potential target of checkpoint control in human cells.

https://science.sciencemag.org/content/sci/277/5331/1501.full.pdf

Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216.

E2F-1, a member of the E2F transcription factor family, contributes to the regulation of the G1-to-S phase transition in higher eukaryotic cells. E2F-1 forms a heterodimer with DP-1 and binds to several cell cycle regulatory proteins, including the retinoblastoma family (RB, p107, p130) and cyclin A/CDK2 complexes. We have analyzed E2F-1 phosphorylation and its interaction with cyclin A/CDK2 complexes both in vivo and in vitro. In vitro, E2F-1 formed a stable complex with cyclin A/CDK2 but not with either subunit alone. DP-1 did not interact with cyclin A, CDK2, or the cyclin A/CDK2 complex. While the complex of cyclin A/CDK2 was required for stable complex formation with E2F-1, the kinase-active form of CDK2 was not required. However, E2F-1 was phosphorylated by cyclin A/CDK2 in vitro and was phosphorylated in vivo in HeLa cells. Two-dimensional tryptic phosphopeptide mapping studies demonstrated an overlap in the phosphopeptides derived from E2F-1 labeled in vitro and in vivo, indicating that cyclin A/CDK2 may be responsible for the majority of E2F-1 phosphorylation in vivo. Furthermore, an active DNA-binding complex could be reconstituted from purified E2F-1/DP-1 and cyclin A/CDK2. Binding studies conducted both in vitro and in vivo demonstrated that the cyclin A/CDK2-binding region resided within the N-terminal 124 amino acids of E2F-1. Because the stable association of E2F-1 with cyclin A/CDK2 in vitro and in vivo did not require a DP-1- or RB-binding domain and because the interactions could be reconstituted from purified components in vitro, we conclude that the interactions between cyclin A/CDK2 and E2F-1 are direct. Finally, we report that the DNA-binding activity of the E2F-1/DP-1 complex is inhibited following phosphorylation by cyclin A/CDK2.

Cyclin A/CDK2 binds directly to E2F-1 and inhibits the DNA-binding activity of E2F-1/DP-1 by phosphorylation.

Cdc25C is a dual-specificfty protein kinase that controls entry into mitosis by dephosphorylating Cdc2 on both threonine 14 and tyrosine 15. cdc25c is phosphorylated on sorine 216 throughout interphase but not during mitosis. Serine 216 phosphorylation mediates the binding of 14-3-3 protein to cdc25C, and Cdc25C/14-3-3 complexes are present throughout interphase but not during mitosis. Here we report the cloning of a human kinase denoted c-TAKIfordc twenty-five Cssociated protein jcinase) that phosphorylates Cdc25C on serine 216 In vitro. C-TAKI Is ubiquitously expressed in human tissues and cell lines and is distinct from the DNA damage checkpoint kinase Chkl , shown previously to phosphorylate Cdc25C on serine 216. Cotransfectlon of Cdc25C with C-TAKI resulted In enhanced phosphorylation of Cdc25C on serine 216. In addition, a physical interaction between C-TAKI and Cdc25C was observed upon transient overexpression in COS-7 cells. Finally, coproduction of Cdc25C and c-TAKI in bacteria resulted in the stoichiometric phosphorylation of Cdc25C on serine 216 and facilitated 14-3-3 protein binding in vitm. Taken together, these results suggest that one function of c-TAKI may be to regulate the interactions between Cdc25C and 14-3-3 in vivo by phosphorylating Cdc25C on serine 216.

http://cgd.aacrjournals.org/cgi/reprint/9/3/197.pdf

C-TAK1 protein kinase phosphorylates human Cdc25C on serine 216 and promotes 14-3-3 protein binding.

In cells of higher eukaryotic organisms the activity of the p34cdc2/cyclin B complex is inhibited by phosphorylation of p34cdc2 at two sites within its amino-terminus (threonine 14 and tyrosine 15)...

/pdf/cell-cycle-regulation-of-the-p34cdc2-inhibitory-kinases-2c3lut44yf.pdf

Cheng Yuan Peng

Papers

Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216.

Cyclin A/CDK2 binds directly to E2F-1 and inhibits the DNA-binding activity of E2F-1/DP-1 by phosphorylation.

C-TAK1 protein kinase phosphorylates human Cdc25C on serine 216 and promotes 14-3-3 protein binding.

Cell cycle regulation of the p34cdc2 inhibitory kinases.

Cyclin A/CDK2BindsDirectly toE2F-1andInhibits the DNA-Binding Activity ofE2F-1/DP-1 byPhosphorylation