BIOLOGICAL OVERVIEW

In response to DNA damage, eukaryotic cells use a system of checkpoint controls to delay cell-cycle progression. Checkpoint delays provide time for repair of damaged DNA before its replication in S phase and before segregation of chromatids in M phase. The Chk2 tumor-suppressor protein has been implicated in certain checkpoint responses in mammalian cells. It directly phosphorylates and inactivates the mitosis-inducing phosphatase Cdc25 (see Drosophila String) in vitro and is required to maintain the G2 arrest that is observed in response to gamma-irradiation. Chk2 also directly phosphorylates p53 (see Drosophila p53) in vitro at a site that is implicated in its stabilization, and is required for stabilization of p53 and induction of p53-dependent transcripts in vivo upon gamma-ionizing radiation. Thus, Chk2 functions in both the G1 and G2 checkpoint responses. Like Chk2, the checkpoint protein kinase ATM (ataxia-telangiectasia-mutated) is required for correct operation of both the G1 and G2 damage checkpoints. ATM is necessary for phosphorylation and activation of Chk2 in vivo and can phosphorylate Chk2 in vitro (Xu, 2001; Abdu, 2002; Masrouha, 2003; and references therein).

The Drosophila serine/threonine kinase Loki (Entree Nucleotide record), here referred to alternatively as Dmnk (Oishi, 1998) or Drosophila Chk2, is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses led to the conclusion that, in flies, Chk2 is required for DNA damage-mediated cell cycle arrest and apoptosis (Xu, 2001). chk2 acts during early embryogenesis to monitor double-strand breaks (DSBs) caused by irradiation during S and G2 phases (Masrouha, 2003). Abdu (2002) presents convincing evidence that chk2 is part of a meiotic checkpoint functioning during oogenesis. The target of this signal is thought to be Vasa, which in turn regulates the translation of Gurken mRNA. Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. Since mutations in human chk2 have been linked to several cancers, these similarities underscore the usefulness of the Drosophila model system (Masrouha, 2003).

In the mammalian system, Chk2 is a key player in maintaining the genome integrity. In the G1 checkpoint, ionizing radiation (IR) exposure can activate the ATM-Chk2 pathway. Activated Chk2 phosphorylates Ser123 in Cdc25A, targeting it for ubiquitin-dependent degradation (Falck, 2001). Since Cdc25A is downregulated, the activity of CyclinA-Cdk2 is inhibited and replication is slowed. In addition, downregulation of Cdc25A is thought to inhibit the activity of CyclinE-Cdk2, leading to the p53-independent initiation of the G1 checkpoint (Bartek, 2001a). In the G2/M checkpoint, Chk1 (Drosophila homolog: Grapes), Chk2, and p53 are three key transducers: the ATM and Rad 3-related (ATR)-Chk1 pathway is thought to be activated when cells are exposed to IR during G1 or S phase; the ATM-Chk2 pathway is thought to arrest cells in response to genotoxic insults during G2 phase (Abraham, 2001); recent findings indicate that p53 may play additional roles to help cells arrest at the G2/M transition (Taylor, 2001). Checkpoint signals relayed from Chk1, Chk2, and p53 arrest the cell cycle at the G2/M transition via downregulation of the kinase activity of CyclinB-Cdk1. Depending on the transducer, this downregulation step can involve one of the following intermediates: Cdc25C, p21, Gadd45, or 14-3-3 (Masrouha, 2003 and references therein).

ATM activates human Chk2 (hChk2) by phosphorylating an amino terminal Thr residue (Thr 68) (Ahn, 2000; Melchionna, 2000), and hChk2, in turn, phosphorylates p53 on two sites, Serine 15 and Serine 20. The location of Ser 15 at the p53 amino terminus suggested that modification of this residue might trigger the dissociation of p53 from MDM2, a protein that targets p53 for ubiquitination, nuclear export, and proteosomal degradation. Therefore, if the model were correct, ATM/ATR-dependent phosphorylation of Ser 15 would free p53 from its destabilizing binding partner, thereby favoring p53 accumulation. It turns out, however, that Ser 15 phosphorylation is not sufficient to disrupt the p53-MDM2 interaction; rather, this modification stimulates the transactivating function of p53 by enhancing the binding of this protein to the transcriptional coactivator, p300. However, these results do not rule out the possibility that phosphorylation of p53 at Ser 15 sets this protein up for a secondary modification that does modify the binding of MDM2 to p53, thereby inhibiting p53 degradation. Indeed, Ser 15 phosphorylation greatly enhances the subsequent phosphorylation of p53 at Ser 18 by casein kinase I, at least under test-tube assay conditions with purified proteins. The presence of phosphates at Ser 15 and Ser 18 reduces the avidity of full-length p53 for MDM2 by approximately threefold. Further studies are required to determine whether, and under what conditions, the tandem modification of p53 by ATM/ATR and casein kinase I contributes to p53 accumulation in intact cells. Chk2 phosphorhylates yet another amino-terminal Ser residue (Ser 20) in p53 (Chehab, 2000; Hirao, 2000; Shieh, 2000). Unlike the Ser 15 modification of Chk2 by ATM, phosphorylation at Ser 20 interferes directly with the binding of p53 to MDM2, thereby favoring p53 accumulation in response to IR-induced DNA damage. The physiological relevance of hChk2 in the regulation of p53 is supported by the finding that loss-of-function mutations in hChk2 can give rise to a variant form of Li-Fraumeni syndrome (Bell, 1999), a heritable, cancer-prone disorder typically associated with germ-line mutations in p53 (Abraham, 2001 and references therein).

In Drosophila, Chk2 appears to act to regulate the apoptotic activity of p53 during genotoxic stress. The tumor suppressor function of p53 has been attributed to its ability to regulate apoptosis and the cell cycle. In mammals, DNA damage, aberrant growth signals, chemotherapeutic agents, and UV irradiation activate p53, a process that is regulated by several posttranslational modifications. Overexpression of Drosophila p53 (p53) in the eye induces apoptosis, resulting in a small eye phenotype. This phenotype is markedly enhanced by coexpression with Drosophila Chk2 and was almost fully rescued by coexpression with a dominant-negative (DN), kinase-dead form of Chk2. DN Chk2 also inhibits p53-mediated apoptosis in response to DNA damage, whereas overexpression of Grapes (Grp), the Drosophila Chk1-homolog, and its DN mutant has no effect on p53-induced phenotypes. Chk2 also activates the p53 transactivation activity in cultured cells. Mutagenesis of p53 amino terminal Ser residues reveals that Ser-4 is critical for its responsiveness toward Chk2. Chk2 activates the apoptotic activity of p53 and Ser-4 is required for this effect. Contrary to results in mammals, Grapes, the Drosophila Chk1-homolog, is not involved in regulating p53. Chk2 may be the ancestral regulator of p53 function (Peters, 2002).

Since maternal Chk2 protein expression persists into embryonic development, the gene may function during early embryogenesis; it was of interest to determine whether chk2 functions in DNA damage checkpoint activation in 3- to 4-hr-old embryos, which are in cycle 14. During cycle 14, cells are known to enter mitosis as stereotypical clusters called 'mitotic domains'. The timing of entry into mitosis of each one of these domains as well as the morphogenetic movements that comprise gastrulation are known to be invariant between different embryos. It was furthermore observed that DNA damage induced by irradiation or MMS treatment can delay entry into mitosis of cycle 14 (Su, 2000); this delay is primarily due to inhibitory phosphorylation of Cdk1, and nuclear exclusion of the Cdk1-Cyclin complex might also play a secondary role (Masrouha, 2003).

Embryos in interphase 14 (130- to 200-min-old embryos) were exposed to 600 rad of gamma-irradiation, which corresponds to the half-lethal dose. Irradiated embryos were allowed to recover for 45 min, after which they were fixed and stained for the mitotic-specific phospho-Histone3 (PH3) epitope. At the same time these embryos were stained for Vasa, a pole-cell-specific marker that shows the progression of the morphogenetic movements of gastrulation. In nonirradiated wild-type embryos, domain 1 initiates mitosis in stage 6. In wild-type embryos, irradiation causes a delay of entry into mitosis. For instance, domain 1 does not start mitosis until much later after irradiation (stage 8). In nonirradiated chk2^null mutant embryos, mitotic patterns in each specific gastrulation stage are the same as in the nonirradiated wild-type embryos. However, in irradiated chk2^null mutant embryos, each mitotic domain enters mitosis with the same timing as the nonirradiated control, indicating that the DNA damage checkpoint is defective. Similar defects in arresting the cell cycle were observed in irradiated chk2^null embryos that were allowed to recover for only 15 min after the gamma-ray exposure. Since S and G2 phases of cycle 14 last 50 and 20 min, respectively, this finding shows that chk2 is a damage checkpoint gene involved in mediating responses to DSBs induced during the S or G2 phases of the cell cycle. Thus, chk2 is required for the DNA damage checkpoint in somatic cells. It thus appears that low Chk2 levels are sufficient for this checkpoint to be active (Masrouha, 2003).

In S. cerevisiae, the Chk2 family member Rad53p is required for the DNA damage and replication checkpoint and arrests the cell cycle at the G1/S transition, in S phase, or at the metaphase-anaphase transition in response to stresses. Nevertheless, Rad53p is not required for the meiotic pachytene checkpoint. Instead, a meiotic-specific version, Mek1p, is required for detecting DNA DSBs that arise as recombination occurs. In S. pombe, the Chk2 family member, Cds1, is mainly required for the S-phase DNA damage/replication checkpoint. Activated Cds1 arrests cells in S phase in response to unreplicated DNA or damaged DNA sensed during S phase. Whether Cds1 is required for the meiotic checkpoint is not yet known. Mammalian Chk2 is indispensable for G1/S, S, and G2/M checkpoint controls, but its role in the meiotic checkpoint is not clear. These functional and temporal divergences between the different CHK2 orthologs indicate that this protein kinase family displays an amazing degree of evolutionary plasticity (Meier, 2001). This plasticity is further supported when one compares C. elegans and Drosophila chk2. While the former has been shown to be required for meiotic chromosome pairing but is dispensable for typical DNA damage/replication checkpoint responses induced by gamma-irradiation or by HU, the results presented in the Masrouha study (2003) show that chk2 has no essential function in Drosophila meiosis. It is involved, however, in monitoring DSBs induced by gamma-rays, which places it closer to its vertebrate homologs and makes it an excellent invertebrate model for studying human chk2 function (Masrouha, 2003).

Calling some of these findings into doubt is a study by Abdu (2002), dealt with in more detail in the Effects of Mutation section. Abdu (2002) presents convincing evidence that chk2 transduces the DSB signal. The target of this signal is thought to be Vasa, which in turn regulates the translation of Gurken mRNA. Resolving this contradiction will require a number of follow-up experiments (Masrouha, 2003).

GENE STRUCTURE

cDNA clone length - 2059 (short form)

Bases in 5' UTR - 270

Exons - 6

Bases in 3' UTR - 409

PROTEIN STRUCTURE

Amino Acids - 459 and 476 (long form)

Structural Domains

A phylogenetic analysis was performed with Loki and its most similar sequences. The Loki polypeptide sequence identified 48 sequences with considerable identity (>25%) in the NCBI databases using the BLAST algorithm. The most conserved sequence, the kinase domain, was then used to perform a multiple alignment that served to generate the phylogenetic tree. The neighbor-joining phylogeny produced a high percentage (94%) branched clade containing Loki, Chk2, Mek1p, Rad53p, Cds1, and Dun1p. In addition, Loki contains a FHA domain (52-112 aa) followed by a kinase domain (157-424 aa), which is the distinguishing feature of Rad53p, Mek1p, Dun1p, Cds1, and Chk2. It is thus likely to have a similar checkpoint function in flies as its homologs in their respective organisms (Masrouha, 2003).

date revised: 8 June 2003

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