Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi


Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101557


Historical Background

From 1970s to early 1990s, a lot of cell-cycle regulators such as cell division cycle (cdc) or radiation-sensitive (rad) genes had been identified as results of yeast genetic screenings. In 1988, through the analyses of rad9 mutants in budding yeast (Saccharomyces cerevisiae), Weinert and Hartwell first proposed a cell cycle checkpoint that prevents inappropriate progression of the cell cycle when DNA is damaged or incompletely replicated (Weinert and Hartwell 1988). In 1993, Beach and coworkers reported a novel gene that controls G2/M transition after DNA damage (G2/M checkpoint) in the fission yeast (Schizosaccharomyces pombe). Since this gene encoded a Ser/Thr protein kinase, it was named checkpoint kinase 1 (Chk1) (Walworth et al. 1993). By 1997, Chk1 orthologs were identified in budding yeast (Rad27), fruit fly (Grapes [Grp]; Drosophila melanogaster), mouse, and human (CHEK1 indicates a gene locus of human Chk1). Chk1 is an evolutionarily conserved kinase involved in the checkpoint signaling (Zhang and Hunter 2014).

Other families of protein kinases were also reported to participate in DNA-damage response (DDR). In budding yeast, Elledge and colleagues reported that Rad53, an ortholog of mammalian checkpoint kinase 2 (Chk2), plays important roles in the checkpoint signaling (Allen et al. 1994). Independently, through the analyses of ataxia-telangiectasia (A-T) patients exhibiting radiation-sensitivity etc., an ataxia-telangiectasia mutated (ATM) gene was reported to encode a phosphatidylinositol 3-kinase (PI3K)-like protein kinase (Savitsky et al. 1995). Elledge’s group also found that TEL1, a budding yeast ortholog of ATM, functions upstream of Rad53 (Sanchez et al. 1996). Rad3, another PI3K-like kinase, was reported to function upstream of Chk1 in fission yeast (Walworth and Bernards 1996). In the same year, an ortholog of Rad3 was identified in mammals and named ATR (ATM- and Rad3-related) (Bentley et al. 1996; Cimprich et al. 1996). Nowadays, ATM-Chk2 and ATR-Chk1 are recognized as kinase cascades critical for DDR (Fig. 1).
Chk1, Fig. 1

Overview of two major DNA-damage response (DDR) pathways in mammalian cells. DNA double-strand break (DSB) initially activates ATM. When chromosomes are already replicated (during S or G2 phase), DNA is resected at the 5′-end of double-strand break (DSB). This DNA structure activates ATR and is required for homologous recombination (HR) repair using the intact sister chromatid as a repair template.

Cell-cycle progression is regulated by the activities of Cyclin-dependent kinases (CDKs). Several sets of Cyclins and CDKs are characterized in mammals: Cyclin B and CDK1 complex is critical for the progression from G2 to M phase (mitosis) and several mitotic events from prophase to metaphase. After a CDK forms a complex with a Cyclin, it is phosphorylated not only on an activating site (e.g., human CDK1-Thr161) by CDK-activating kinase (CAK) but also on two neighboring inhibitory sites (e.g., human CDK1-Thr14 and -Tyr15) by Wee1 and/or Myt1 (Fig. 1). Cdc25 protein phosphatases finally remove these inhibitory phosphates and then activate CDKs (Fig. 1): three Cdc25 isoforms (A-C) exist in human. In 1997, Russell and coworkers first demonstrated that Chk1 phosphorylates and inhibits Cdc25, whereas Wee1 is not a main target of Chk1 in fission yeast (Furnari et al. 1997). Simultaneously, Piwnica-Worms and colleagues also demonstrated that Chk1 is able to phosphorylate Cdc25C at a conserved serine residue (human Cdc25C at Ser216) (Peng et al. 1997). However, since the same group reported that human Cdc25-Ser216 is phosphorylated mainly by C-TAK1 and the level of its phosphorylation does not dramatically change after DNA damage (Peng et al. 1998), the role of Cdc25C phosphorylation in DDR has been a matter of debate (Goto et al. 2012). Later, the group of Bartek and Lukas demonstrated that Chk1 triggers Cdc25A degradation (Mailand et al. 2000). Nowadays, with regard to cell-cycle arrest, Cdc25A is considered as the most important target of Chk1. In subsequent chapters, we will explain Chk1 regulation and function with a particular focus on a human ortholog.

Chk1 Function in DNA-Damage Response (DDR)

DNA is constantly being damaged exogenously (e.g., by ultraviolet [UV], ionizing radiation [IR], or chemicals including anticancer drugs) or endogenously (e.g., by free radicals or by-products of intracellular metabolism). These DNA alterations activate DNA damage checkpoint so that cells do not enter next cell cycle until damaged DNA is repaired. ATM-Chk2-p53 and ATR-Chk1-Cdc25A are two major routes to suppress CDK activities and then arrest cell cycle at a certain phase (G1/S, intra-S, or G2/M).

ATM activation is primarily induced by DNA double-strand break (DSB) or oxidative stress. DSB can be generated by IR or some chemicals such as topoisomerase inhibitors (Fig. 1). ATM phosphorylates and activates Chk2. Activated ATM and Chk2 phosphorylate p53, resulting its stabilization. The stabilized p53 stimulates the transcription of p21, one of CDK inhibitors (CKIs). CDK activities are inhibited by the binding of Cyclin and CDK complexes to p21, which results in cell-cycle arrest.

On the other hand, ATR activation requires the generation of single-strand DNA (ssDNA) adjacent to double-strand DNA (dsDNA). This DNA structure is generated during the stalling of DNA replication or the processes of DNA repair, such as homologous recombination (HR) repair (e.g., DSB repair only when intact replicated DNA exists [during S or G2 phase]), nucleotide exclusion repair (NER; e.g., the repair after UV irradiation), or inter-strand crosslink (ICL) repair (e.g., the repair after the treatment with cisplatin; Fig. 1). The activated ATR phosphorylates Ser/Thr residues prior to Gln (SQ and TQ sequences) on several target substrates, corresponding to Ser317 and Ser345 on human Chk1 (Fig. 2).
Chk1, Fig. 2

Overview of functional changes of Chk1 by phosphorylation during DDR

ATR-induced Chk1 phosphorylation increases the catalytic activity of Chk1 but the elevation is only modest (Zhou and Elledge 2000). In addition to the catalytic activation, the phosphorylation triggers the change in subcellular localization of Chk1 (Fig. 2). In mammals, Chk1 is accumulated in the nucleus after DNA damage (Sanchez et al. 1997). Nuclear export signal (NES) sequence exists near Ser345 on Chk1, the phosphorylation of which creates a docking site for 14-3-3 β or ζ (Jiang et al. 2003). The binding to these 14-3-3 proteins shields Chk1-NES from Crm-1 (a protein required for nuclear export; also named exportin 1), resulting in nuclear retention of Chk1 (Jiang et al. 2003). Since nuclear Chk1 activity is essential to establish a checkpoint (Reinhardt et al. 2010; Matsuyama et al. 2011), this nuclear accumulation also facilitates Chk1 function. The phosphorylation by ATR also induces Chk1 autophosphorylation at several sites including Ser296. Once Chk1 is autophosphorylated, ATR sites are rapidly dephosphorylated (Kasahara et al. 2010). The shift in Chk1 phosphorylation sites coincides with the translocation of Chk1 from chromatin (DNA damage foci) to entire nucleus (Kasahara et al. 2010), which enables Chk1 to deliver checkpoint signals throughout the nucleus.

Among Chk1 autophosphorylation sites, Chk1-Ser296 is the most critical site to arrest cell cycle after DNA damage (Fig. 2). Ser296 autophosphorylation enables Chk1 to associate with 14-3-3 in a γ-subtype-specific manner (Kasahara et al. 2010). During DDR, Chk1 also creates 14-3-3-docking sites on Cdc25A through its phosphorylation at Ser178 and Thr507 (Chen et al. 2003). Since 14-3-3 proteins form homo- and hetero-dimers, these phosphorylations by Chk1 facilitate the complex formation between Chk1 and Cdc25A on 14-3-3 proteins (Kasahara et al. 2010). This complex formation is critical for Chk1 to phosphorylate Cdc25A at Ser76 (Kasahara et al. 2010), a rate-limiting phosphorylation site for Cdc25A degradation. Thus, Chk1-Ser296 autophosphorylation occurs after ATR-induced phosphorylation and is important to transduce checkpoint signals to Cdc25A (Goto et al. 2012, 2015).

In this chapter, we have focused on main checkpoint signaling pathways to induce cell-cycle arrest during DDR. A lot of other proteins have been identified as target substrates for ATM, ATR, Chk1, or Chk2. Readers are also referred to recent reviews (Reinhardt and Yaffe 2009; Shiloh and Ziv 2013; Zhang and Hunter 2014; Awasthi et al. 2015) or public databases such as PhosphoSitePlus® (http://www.phosphosite.org).

Chk1 Function in Cell Viability

DNA damage checkpoint pathways were initially considered to function only in response to exogenously introducing DNA-damaging stresses. However, accumulating evidence has suggested that ATR-Chk1-Cdc25A pathway plays critical roles in cell viability under physiological conditions at least in mammals. The fact that each gene knockout mouse died in early embryogenesis is one example that illustrates the importance of ATR-Chk1-Cdc25A pathway in unperturbed cells (Zhou and Elledge 2000; Goto et al. 2012; Zhang and Hunter 2014). In the following, we will focus on Chk1 function under physiological conditions without exogenously introducing DNA-damaging stresses.

Chk1 activity is required for both the prevention of late origin firing and the elongation of replication fork, the disturbance of which results in irreversible replication fork collapse (Zhang and Hunter 2014). Thus, Chk1 likely plays critical roles in the management of DNA replication during S phase (Fig. 3). In addition to S phase function, Chk1 negatively regulates cell-cycle progression (especially from G2 to M phase) in unperturbed cells (Zhang and Hunter 2014). However, there is a debate as to whether Chk1 constitutively exhibits a catalytic activity to some extent in the absence of DNA damage or reacts with spontaneous DNA damages (induced by free radicals, etc.) in normal cell-cycle progression.
Chk1, Fig. 3

Chk1 localization in a cell-cycle-dependent manner. During the S phase, Chk1 inhibits the firing of late replication origin, stabilizes replication fork, and elongates replicated DNA. Without exogenous DNA-damaging stresses, Chk1 negatively regulates cell-cycle progression likely through the inhibition of CDK activities in the nucleus. Chk1 localization is regulated by several kinases, such as p90RSK and CDK1

Since Chk1 has an activity to restrain cells in the G2 phase, this activity has to be invalidated at the G2/M transition. Once CDK1 is activated at the G2/M boundary, it starts to phosphorylate Chk1 at Ser286 and Ser301 (Fig. 3). This phosphorylation facilitates Chk1 binding to Crm-1, which accelerates Chk1 export from nucleus to cytoplasm. Chk1 exclusion from nucleus relieves its inhibitory effects on nuclear CDK1 activity, which promotes mitotic progression (Enomoto et al. 2009; Matsuyama et al. 2011).

Chk1 is localized diffusely in the nucleus and cytoplasm during quiescent state (at the G0 phase), whereas Chk1 is localized in the nucleus more than in the cytoplasm during proliferation state (from G1 to G2 phase; Fig. 3). This nuclear accumulation of Chk1 is controlled by Chk1-Ser280 phosphorylation, which is governed by p90RSK (ribosomal S6 kinase) downstream of MAP kinase cascade (Li et al. 2012). Since the disturbance of Chk1-Ser280 phosphorylation results in the delay of DDR (Li et al. 2012), Chk1 likely monitors genomic integrity at the nucleus throughout proliferation state.

Chk1 in Cancer

Normal cells have two major DDR pathways, ATM-Chk2-p53 and ATR-Chk1-Cdc25A. Hereditary mutations were reported at ATM, CHEK2, and TP53 gene loci in patients with cancer preposition syndromes. The fact that homozygous impairments of these genes were frequently observed in a variety of sporadic human cancers is another example that illustrates the importance of ATM-Chk2-p53 pathway as tumor suppressor (Goto et al. 2012). On the other hand, no homozygous loss-of-function mutations have been detected in CHEK1 gene locus of cancer specimens, whereas its heterozygous deletion was reported in a few types of cancers. Rather, Chk1 had been reported to be upregulated in a variety of cancers (Zhang and Hunter 2014). The impairment of ATM-Chk2-p53 pathway likely leads to the upregulation of ATR-Chk1-Cdc25A pathway in a majority of cancer cells. Based on these backgrounds, several pharmaceutical companies are trying to establish Chk1 inhibitors as molecular target drugs for an anticancer therapy.

Since Chk1 upregulation is well correlated with tumor grade and recurrence in some types of cancers, it may be associated with the resistance of DNA-damaging therapies, such as radiotherapy or conventional chemotherapy (Zhang and Hunter 2014). Thus, Chk1 inhibitors were initially expected to sensitize cancer cells to conventional anticancer therapies (Goto et al. 2012). However, the use of several drugs likely increases the risk of undesirable effects, such as their off-target effects and toxicity. So, more attention has been paid to single use of Chk1 inhibitor. In preclinical models, cancer cells are more sensitive to Chk1 inhibitors than normal cells (Goto et al. 2015). This phenomenon may reflect more replicative stresses and/or endogenous DNA-damaging stresses (induced by free radicals or by-products of intracellular metabolism) in cancer cells. However, the precise mechanism by which Chk1 inhibitors preferentially kills cancer cells is largely unknown.


Cell cycle checkpoints primarily work to induce cell-cycle arrest, which provides the time to repair DNA damages. Checkpoint kinase 1 (Chk1) is an evolutionally conserved protein kinase to transduce checkpoint signals from ATR to Cdc25 during DNA-damage response (DDR). In mammalian cells, Chk1 is also critical for cell viability under physiological conditions without exogenously introducing DNA-damaging stresses. Chk1 is likely to manage DNA replication during S phase and negatively regulate cell-cycle progression especially from G2 to M phase, but the precise mechanism(s) remain largely unknown. Since Chk1 is generally upregulated in cancer and critical for cancer cell survival, Chk1 is now considered as one of the promising molecular targets for anticancer therapy. The development of Chk1-targeted therapy calls for a more advanced molecular understanding of Chk1 especially in normal cell-cycle progression. At the end, we sincerely apologize for being unable to cite all related publications due to space limitations.


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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Division of BiochemistryAichi Cancer Center Research InstituteNagoyaJapan
  2. 2.Department of Cellular Oncology, Graduate School of MedicineNagoya UniversityNagoyaJapan
  3. 3.Department of PhysiologyMie University School of MedicineTsuJapan