Chk2 activates the apoptotic activity of p53

The tumor suppressor function of p53-1 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. In Drosophila, however, the regulation modes of p53 are still unknown. Overexpression of Drosophila 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 revealed 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).

Various forms of cellular stress such as DNA damage or ionizing irradiation lead to activation and stabilization of the p53 tumor suppressor protein and to growth arrest and apoptosis. Chk2, the mammalian homolog of the Saccharomyces cerevisiae Rad 53 and the Schizosaccharomyces pombe Cds1 checkpoint genes, regulate p53 function in mammals in response to DNA damage (Chehab, 2000). Chk2 is a protein kinase that acts downstream of the ataxia telangiectasia mutated (ATM) kinase and may induce cell cycle arrest (Matsuoka, 1998; Blasina, 1999). Loss of Chk2 in thymocytes results in failure to increase intracellular p53 levels in response to DNA damage, causing a defect in p53-mediated apoptosis (Hirao, 2000). Chk1, an evolutionarily conserved protein kinase, implicated in cell cycle checkpoint control in lower eukaryotes (Rhind, 2000: Rhind, 1998; Russell, 1998), also has been suggested to play a role in p53 regulation (Walworth, 2000; Zhou, 2000). Chk1 also can phosphorylate p53, probably at the same sites as Chk2 (Shieh, 2000). Currently, the relative significance of p53 phosphorylation by Chk2 and/or Chk1 in the process of p53 activation is unclear. Several components involved in cell cycle checkpoint control pathways are conserved in Drosophila. Drosophila homologs of the ATM/ATR (mei-41: Hari, 1995), Chk1 (grapes), and Chk2 (loki; Oishi, 1998) kinases have been identified. Mei-41 mutant cells are sensitive to ionizing radiation, display high levels of mitotic chromosome instability, and do not arrest upon radiation treatment (Hari, 1995). Grapes (Grp) has been shown to be involved in a developmentally regulated DNA replication/damage checkpoint operating during the late syncytial divisions. The Drosophila maternal nuclear kinase (DMNK) protein is the homolog of the human Chk2 protein [referred to as Drosophila melanogaster Chk2 (Chk2)] and is highly expressed in Drosophila ovaries and functions in meiosis (Oishi, 1998). The role of Chk2 or Grp in the regulation of Drosophila p53 (mp53) is unknown. It is also not clear, as to whether Chk2 functions in a cell cycle checkpoint pathway in vivo. Data from C. elegans suggest that Chk2 mutants are defective in meiosis but retain a DNA damage checkpoint in response to replication inhibition and ionizing radiation (Higashitani, 2000; MacQueen, 2001). Cloning and characterization of Drosophila p53 has shown an essential role for Dmp53 in radiation-induced apoptosis. Overexpressing of Drosophila p53 in the fly eye results in massive apoptosis and a small eye phenotype. These results have now been expanded by using genetic epistasis experiments; an essential role has been demonstrated for Drosophila Chk2 in the regulation of p53. The amino-terminal Ser at position 4 of the p53 molecule confers responsiveness toward Chk2. Interestingly, Grp is not involved in the regulation of Dmp53 in this system (Peters, 2002 and references therein).

Overexpression of human p53 in the Drosophila eye results in a striking reduction in eye size and a disruption of the ommatidia structure. Ommatidia are fused and exhibit a complete loss of bristles. Overexpression of Drosophila p53 produces a less severe phenotype, resulting in a reduced eye size with partial fusion of the ommatidia and some remaining bristles. Consistent with the known activity of the GMR promoter, the expression of both proteins was detected in the eye imaginal disc posterior to the morphogenetic furrow (Peters, 2002).

Expression of Drosophila or human p53 resulted in extensive apoptosis in the developing eye, as judged by acridine orange staining. The S-phase band posterior to the morphogenetic furrow, as visualized by BrdUrd immunohistochemistry, is present in both human p53 and Drosophila p53 transgenic flies. No considerable differences in cell cycle distribution or cell size could be discerned when human p53 or Drosophila p53-overexpressing flies were compared. These results show that overexpression of either human or Drosophila p53 resulted in apoptosis in the developing fly eye without detectable effects on cell cycle regulation (Peters, 2002).

To study the interaction between Chk2 and p53, the cDNA encoding the Drosophila homolog of human Chk2 was cloned. A kinase-dead form of Drosophila Chk2 was generated, in which the conserved aspartic acid at position 303 was mutated to Ala (DN-Chk2). Mutation of this amino acid has been shown (Matsuoka, 1998) to function as a DN mutation (Peters, 2002).

Drosophila Chk2 and DN-Chk2 were overexpressed in the Drosophila eye, under the control of the GMR promoter. No overt phenotype could be observed and no apoptotic cells were detected in the eye imaginal discs from third-instar larvae (Peters, 2002).

To study the genetic interaction between Drosophila p53 and Chk2, transgenic flies overexpressing p53 were crossed with transgenic flies overexpressing either wild-type or DN-Chk2. Coexpression of wild-type Chk2 and p53 results in a considerably more severe phenotype with almost complete loss of the eye compared to flies expressing p53 alone. Excessive apoptosis was detected in eye imaginal discs from third-instar larvae coexpressing Chk2 and p53. In contrast, coexpression of DN-Chk2 with p53 results in an almost complete rescue of the Drosophila p53-induced eye phenotype. Consistent with the observed eye morphology, no apoptosis could be found in eye imaginal discs in these animals. Analysis of p53 protein levels by immunohistochemistry in flies coexpressing wild-type or DN-Chk2 and p53 revealed no difference compared to flies expressing p53 alone, indicating that altered p53 protein levels do not account for the observed changes in eye phenotype (Peters, 2002).

A kinase assay was performed to confirm Drosophila Chk2 activity. Chk2 phosphorylates a synthetic Chk1/Chk2 peptide substrate. In the presence of increasing amounts of DN-Chk2, the kinase activity of the wild-type Chk2 is lost. These experiments show that the D303A mutant of Chk2 functions as a true DN protein, inhibiting the kinase activity of the wild-type protein (Peters, 2002).

Drosophila p53-mediated sensitivity to irradiation was investigated in wild-type and transgenic flies overexpressing a DN form of p53 (p53-D259H). The D259H point mutation corresponds to the human p53 mutational hotspot at position 273. In human p53, this amino acid directly contacts DNA and is required for DNA binding. Eye imaginal discs were dissected from third-instar larvae 4 h after gamma-irradiation, and apoptotic cells were visualized with acridine orange. Wild-type, unirradiated eye discs do not show apoptotic cells. In contrast, irradiated wild-type eye discs exhibit a high number of apoptotic cells both in the antenna discs and in the anterior and posterior part of the eye imaginal discs. Irradiated eye discs from flies overexpressing p53-D259H show abundant apoptotic cells only anterior to the morphogenetic furrow, where p53-D259H is not expressed. Posterior to the morphogenetic furrow, where p53-D259H is expressed, few apoptotic cells were present. These results show that p53 is implicated in gamma-irradiation-induced apoptosis in the Drosophila eye (Peters, 2002).

Irradiated eye imaginal discs overexpressing wild-type Chk2 posterior to the morphogenetic furrow show high numbers of acridine orange-positive apoptotic cells in this area. However, in DN-Chk2-overexpressing eye discs, there is almost a complete absence of apoptotic cells, comparable to discs overexpressing the DN form of p53. These results demonstrate a role for Chk2 in the regulation of gamma-irradiation-induced apoptosis in the developing eye in Drosophila (Peters, 2002).

The ability of Drosophila p53 to activate transcription in Drosophila S2 cells in the presence of wild-type or DN-Chk2 and Grp was analyzed by using a human p53 responsive CAT-reporter construct (PG13-CAT). Transfection of Drosophila p53 results in a dose-dependent increase in PG13-CAT reporter activity. Cotransfection of Drosophila Chk2 with Drosophila p53 causes a further increase in reporter activity. In contrast, expression of DN-Chk2 interfers with Drosophila p53-mediated transcription. In agreement with genetic studies, wild-type and DN Grp constructs have no influence on p53 transcriptional activity. p53 protein levels were unchanged by cotransfection with either wild-type or DN-Chk2 or Grp constructs, indicating that the observed effects are not caused by changes in Drosophila p53 protein levels. These results establish Chk2, but not Grp, as a regulator of p53 transcriptional activity in Drosophila (Peters, 2002).

Phosphorylation and acetylation have been implicated in regulating mammalian p53 stability and transcriptional activity after DNA damage. Sites of particular interest are the Ser-Glu amino acid pairs at positions 15 and 37 of human p53, which can be phosphorylated on the Ser residues by members of the ATM family of DNA damage-responsive kinases like ATM, Chk1, or Chk2. Based on homology, the nearby Ser-4-Glu-5 pair in Drosophila p53 might be a target for one of these kinases (Peters, 2002).

To define whether Drosophila p53 phosphorylation is part of the mechanism in the regulation of Drosophila p53 by Chk2, several point mutations (Drosophila p53-S4A, -S8A, -S16A, and -S20A) were introduced into the amino-terminal part of p53. Transfection of the various mutant Drosophila p53 molecules resulted in a similar increase in PG13-CAT-reporter activity as observed with the transfection of wild-type p53. Cotransfection of Chk2 with p53-S20A causes a strong increase in reporter activity similar to wild-type p53, whereas expression of DN-Chk2 interfers with Drosophila p53-S20A. Mutation of Ser-8 and Ser-16 also does not interfere with the transcriptional activation of p53 by Chk2 cotransfection. These results suggest that Ser-8, -16, and -20 are not required for the regulation of Drosophila p53 activity by Chk2 in Drosophila (Peters, 2002).

Mutating Ser-4 of p53, however, makes p53 unresponsive to Chk2. When Drosophila p53-S4A is cotransfected with wild-type or DN DmChk2, no increase or decrease of the PG13-CAT-reporter activity is observed; this indicates that Ser-4 might be crucial in the regulation of Drosophila p53 by Chk2. The D259H point mutation does not induce any reporter activity and is not affected by cotransfection with either wild-type or DN DmChk2 (Peters, 2002).

The analysis was extended into the Drosophila eye. Transgenic flies expressing some of the Ser mutants of Drosophila p53 were constructed. Overexpression of the Drosophila p53 mutants S4A and S20A in the fly eye results in a small eye phenotype identical to the overexpression of wild-type Drosophila p53. Genetic epistasis was performed crossing these flies to transgenic flies overexpressing either wild-type or DN-Chk2. In agreement with the results from S2 cells, Drosophila p53-S20A behaves indistinguishably from the wild-type Drosophila p53, whereas the S4A mutant results in a phenotype that is unaltered by coexpression with wild-type or DN-Chk2. These results confirm that Ser-4 of Drosophila p53 is important for its regulation by Chk2; mutating Ser-4 makes p53 unresponsive to Chk2 (Peters, 2002).

Upon DNA damage, cells respond with cell cycle arrest and activation of genes that coordinate DNA repair. If these mechanisms fail, genomic instability and predisposition for the development of cancer is the consequence. p53 is central to the execution of the DNA damage response. Regulation of p53 is coordinated mainly by two mechanisms: regulation of its stability and its activity. Ionizing irradiation induces phosphorylation of several amino-terminal amino acids of mammalian p53, a process that is mediated by both Chk1 and Chk2. Phosphorylation of human p53 at Ser-20 interferes with binding of murine double minute 2 protein (MDM2) to p53, thereby inhibiting degradation of p53 and leading to an increase in p53 protein levels (Chehab, 2000; Hirao, 2000; Shieh, 2000). The mechanism underlying the regulation of p53 transcriptional activity is less clear, but phosphorylation of p53 could be involved in this process as well. In mammals, both regulatory mechanisms are tightly linked together and are difficult to differentiate. In Drosophila, genomewide searches have not identified an MDM2 homolog, allowing the investigation of the mechanism of p53-regulation independent of MDM2 (Peters, 2002).

This study demonstrates that in Drosophila, Chk2 is a potent activator of p53. Expression of both molecules in the Drosophila eye leads to a massive reduction in eye size caused by an increase in the number of apoptotic cells. Interestingly, the requirement for Chk2 to activate p53 is observed only upon overexpression or Drosophila p53 or after radiation treatment, since overexpression of Chk2 or DN-Chk2 alone does not show a phenotype. Xu (2001) also has shown that flies homozygously deleted for Chk2 are viable and lack obvious phenotype in the absence of irradiation. These observations suggest that endogenous p53 may function primarily in a DNA damage response. A kinase-dead, DN-Chk2 is able to almost fully rescue the p53-induced phenotype by inhibiting apoptosis in the eye imaginal discs. A functional interaction between Grp and p53 could not be detected, suggesting that Chk2 is the principal activator of Drosophila p53 in Drosophila. In an attempt to define the molecular mechanism of the observed interaction between p53 and Chk2, several point mutations were introduced into the amino-terminal part of the Drosophila p53 molecule and tested for their responsiveness to wild-type or DN-Chk2. The transcriptional activity and the eye phenotype caused by Drosophila p53-S4A mutant were unaffected by coexpression with either wild-type or DN-Chk2. These results indicate that Ser-4 of Drosophila p53 confers responsiveness to Chk2 (Peters, 2002).

It is hypothesized that the regulation of p53 by Chk2 in Drosophila could be mediated by direct phosphorylation. However, in vitro Chk2 kinase assays using wild-type or mutant glutathione S-transferase-p53 proteins as substrates did not detect any differences in phosphorylation between the various p53 molecules. One explanation for this phenomenon is that Chk2 does not directly phosphorylate Ser-4 and that a Chk2-regulated kinase is involved in this process. Alternatively, Chk2 may phosphorylate p53 at multiple sites, preventing discrimination between wild-type and Drosophila p53-S4A phosphorylation levels (Peters, 2002).

The mutationally altered p53 (p53-S4A) should be insensitive to activation by Chk2; however, p53-S4A overexpressing flies exhibit a phenotype similar to p53 wild-type transgenic flies. This finding is surprising given that DN-Chk2 almost completely rescues the Drosophila p53-induced small eye phenotype. There may be several reasons for this. Other signals not related to Chk2 that may or may not involve phosphorylation could be constitutively active, resulting in p53 stabilization and transcriptional activity. In this case, mutation of S4 could inhibit further activation of Drosophila p53 but would not prevent the constitutive activating signals present. Alternatively, introduction of the S4 mutation could introduce steric changes that confer increased stability to the p53 molecule. The increased stability could also account for the observed phenotype. A further possibility is that Chk2 additionally activates a kinase that phosphorylates Drosophila p53. In this situation, mutation of Drosophila p53 Ser-4 would prevent direct activation of p53 but would not interfere with indirect activation. This also would explain why DN-Chk2 could prevent Drosophila p53-mediated apoptosis (Peters, 2002).

This study has shown that radiation-induced cell death is inhibited by DN-Drosophila p53 and DN-Chk2 and that apoptosis resulting from Drosophila p53 overexpression can be inhibited by a kinase-dead form of Chk2. Chk2 regulation of Drosophila p53 activity is crucial for gamma-radiation-induced apoptosis, consistent with data showing that Chk2 functions upstream of Drosophila p53. In favor for this view, Xu (2001) described Chk2 null flies that are refractory to apoptosis upon irradiation, suggesting that Chk2 controls p53-induced apoptosis. However, a scenario in which p53 and Chk2 make independent contributions to radiation-induced cell death, functioning in separate pathways cannot be ruled out (Peters, 2002).

Since Drosophila most likely does not have an MDM2 gene, the MDM2-mediated p53 degradation pathway could have emerged at a later point in evolution. The data presented here suggest that MDM2-independent regulation of Drosophila p53 is mediated by Chk2 and implies that Drosophila Chk1 does not function in this process. If MDM2-independent regulation of p53 is more ancient in evolutionary terms, then Chk2 may be the ancestral regulator of p53 activation. Thus, MDM2 and Chk1 probably emerged as regulators of p53 at a later evolutionary time (Peters, 2002).

The ability of Chk2 to regulate transcriptional activity of Drosophila p53 suggests that in mammals Chk2 might be involved in control of both p53 stability and activity. Generation of mouse 'knock-in' mutants for individual amino-terminal p53 phosphorylation sites should aid in resolution of apparent multiple roles of Chk2 in p53 regulation (Peters, 2002).

ATM and ATR pathways signal alternative splicing of Drosophila TAF1 pre-mRNA in response to DNA damage

Alternative pre-mRNA splicing is a major mechanism utilized by eukaryotic organisms to expand their protein-coding capacity. To examine the role of cell signaling in regulating alternative splicing, the splicing of the Drosophila TAF1 pre-mRNA was analyzed. TAF1 encodes a subunit of TFIID, which is broadly required for RNA polymerase II transcription. TAF1 alternative splicing generates four mRNAs, TAF1-1, TAF1-2, TAF1-3, and TAF1-4, of which TAF1-2 and TAF1-4 encode proteins that directly bind DNA through AT hooks. TAF1 alternative splicing was regulated in a tissue-specific manner and in response to DNA damage induced by ionizing radiation or camptothecin. Pharmacological inhibitors and RNA interference were used to demonstrate that ionizing-radiation-induced upregulation of TAF1-3 and TAF1-4 splicing in S2 cells is mediated by the ATM (ataxia-telangiectasia mutated) DNA damage response kinase and checkpoint kinase 2 (CHK2), a known ATM substrate. Similarly, camptothecin-induced upregulation of TAF1-3 and TAF1-4 splicing is mediated by ATR (ATM-RAD3 related) and CHK1. These findings suggest that inducible TAF1 alternative splicing is a mechanism to regulate transcription in response to developmental or DNA damage signals and provide the first evidence that the ATM/CHK2 and ATR/CHK1 signaling pathways control gene expression by regulating alternative splicing (Katzenberger, 2006; Full text of article).

valois encodes a divergent WD protein that regulates chk2 translation

valois (vls) was identified as a posterior group gene in the initial screens for Drosophila maternal-effect lethal mutations. Despite its early genetic identification, it has not been characterized at the molecular level until now. vls encodes a divergent WD domain protein and the three available EMS-induced point mutations cause premature stop codons in the vls ORF. A null allele was identified that has a stronger phenotype than the EMS mutants. The vlsnull mutant shows that vls+ is required for high levels of Oskar protein to accumulate during oogenesis, for normal posterior localization of Oskar in later stages of oogenesis and for posterior localization of the Vasa protein during the entire process of pole plasm assembly. There is no evidence for vls being dependent on an upstream factor of the posterior pathway, suggesting that Valois protein (Vls) instead acts as a co-factor in the process. Based on the structure of Vls, the function of similar proteins in different systems and phenotypic analysis, it seems likely that vls may promote posterior patterning by facilitating interactions between different molecules (Cavey, 2005).

chk2 and vls are encoded by opposite strands and cDNA sequence data shows that their 3'UTRs are complementary over 127 nucleotides. chk2 is translationally repressed by orb during oogenesis, and because translational control often relies on the binding of trans-acting factors to sequences in the 3'UTR of mRNAs, it was of interest to know whether vls could also play a role in chk2 translational control. Indeed, Chk2 levels increase about 6-fold in vlsPG65/HC33 and vlsPG65/RB71 ovaries compared with wild type and this is close to the 10-fold upregulation reported for orb mutants. This indicates that vls is also involved in the regulation of Chk2 levels. However, orb does not simply function to control Vls levels because these are normal in orb mutants (Cavey, 2005).

Checkpoint kinase 2 (Chk2) inhibits the activity of the Cdc45/MCM2-7/GINS (CMG) replicative helicase complex

The replication of eukaryote chromosomes slows down when DNA is damaged and the proteins that work at the fork (the replisome) are known targets for the signaling pathways that mediate such responses critical for accurate genomic inheritance. However, the molecular mechanisms and details of how this response is mediated are poorly understood. This report shows that the activity of replisome helicase, the Cdc45/MCM2-7/GINS (CMG) complex, can be inhibited by protein phosphorylation. Recombinant Drosophila CMG can be stimulated by treatment with phosphatase whereas Chk2 but not Chk1 interferes with the helicase activity in vitro. The targets for Chk2 phosphorylation have been identified and reside in MCM subunits 3 and 4 and in the GINS protein Psf2. Interference requires a combination of modifications and it is suggested that the formation of negative charges might create a surface on the helicase to allosterically affect its function. The treatment of developing fly embryos with ionizing radiation leads to hyperphosphorylation of Psf2 subunit in the active helicase complex. Taken together these data suggest that the direct modification of the CMG helicase by Chk2 is an important nexus for response to DNA damage (Ilves, 2012).

Protein Interactions

The Dmnk (Drosophila maternal nuclear kinase, Chk2/loki) gene, encoding a nuclear protein serine/threonine kinase, is expressed predominantly in the germline cells during embryogenesis, suggesting its possible role in the establishment of germ cells. Dmnk interacts physically with Drosophila RNA binding protein Orb, which plays crucial roles in the establishment of Drosophila oocyte by regulating the distribution and translation of several maternal mRNAs. Considering similar spatiotemporal expression patterns of Dmnk and orb during oogenesis and early embryogenesis, it is suggested that Dmnk plays a role in establishment of germ cells by interacting with Orb. Although there are two forms of Dmnk proteins, Dmnk-L (long) and Dmnk-S (short) via the developmentally regulated alternative splicing, Orb can associate with both forms of Dmnk proteins when expressed in culture cells. However, immunohistochemical analysis has revealed that Dmnk-S, but not Dmnk-L, can affect the subcellular localization of Orb in a kinase activity-dependent manner, suggesting differential functions of Dmnk-S and Dmnk-L in the regulation of Orb (Iwai, 2002).

loki: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.