The genomic region containing reaper, grim, and head involution defective is required for all cell death in Drosophila embryos, including radiation-induced apoptosis. rpr is transcriptionally induced in embryos following irradiation, and an 11 kb sequence upstream of the rpr start codon is sufficient to confer radiation responsiveness on a lacZ reporter transgene. To identify the minimal radiation-responsive cis-elements upstream of rpr, the ability of smaller fragments of this 11 kb regulatory region to activate lacZ transcription was tested. Each transgenic strain was tested for radiation-induced expression of beta-galactosidase. Multiple constructs containing sequences ~5 kb upstream of the rpr start codon show a robust radiation response. These experiments identify a discrete 150 bp enhancer that responds to radiation as strongly as the larger enhancer fragments tested. Since this enhancer retains radiation-responsiveness but does not recapitulate the developmental patterns of rpr expression seen with larger enhancer fragments, the results also indicate that cis-regulatory sequences responsible for damage-induced transcription of rpr can be isolated from others that respond to developmental cues (Brodsky, 2000a).
Within the 150 bp enhancer, a 20 bp sequence was identified that strongly resembles the consensus for human p53 DNA-binding sites. This 20 bp sequence is referred to as the p53 response element (p53RE) to reflect its response to Dmp53 in yeast. Like those found upstream of the human target genes mdm-2 and p21/WAF1, this putative p53 binding site upstream of rpr contains two tandemly arrayed 10mers, each of which matches the consensus motif at nine of ten positions. The two mismatches occur at the outer positions of the 20 bp element; the invariant core nucleotides of each 10mer motif match the consensus perfectly (Brodsky, 2000a).
Yeast one-hybrid assays were used to see whether Drosophila p53 interacts with the p53RE. For these studies, a reporter plasmid containing the p53RE upstream of the beta-galactosidase gene was integrated into the yeast genome to produce the p53RE bait strain. Next, this p53RE bait strain was transformed with test plasmids expressing either wild-type Dmp53 or Dmp53(259H) fused to the GAL4 activation domain. These strains were assayed for beta-galactosidase activity. Reporter expression in strains with Dmp53(259H) or the empty vector control (expressing the Gal4 activation domain alone) are indistinguishable from each other. Compared to these controls, each of the four independent transformants carrying the wild-type Dmp53 plasmid shows a substantial increase in beta-galactosidase levels. Based on these results, it has been concluded that the 150 bp radiation-responsive enhancer upstream of rpr contains a 20 bp binding site for Dmp53 (Brodsky, 2000a).
A test was performed to see whether the p53RE is sufficient to confer radiation-responsive transcriptional activation on a lacZ reporter construct in vivo. A transgene containing four copies of the p53RE and the minimal hsp70 promoter has showennegligible expression in untreated embryos but is substantially induced following irradiation. Therefore, the 20 bp Dmp53 binding site from the rpr locus is sufficient to mediate a transcriptional response to radiation and may define a minimal radiation responsive sequence. When analyzed in parallel to the 150-lacZ reporter, containing 150 bases surrounding the p53RE, the p53RE-lacZ reporter exhibits less robust and less uniform beta-galactosidase activity following irradiation. Reduced activity is often observed when DNA elements are tested in isolation from the normal flanking sequences and, in this instance, may reflect the influence of other factors that interact with the 150 bp enhancer sequence (Brodsky, 2000a).
Disruptions of development are often associated with excess apoptosis. For example, in a crumbs (crb) mutant background, abnormal epidermal development in the embryo leads to widespread apoptosis. This apoptosis is fully suppressed by deletions for the genomic region containing rpr, hid, and grim and is preceded by a dramatic induction of rpr expression, similar to that seen in irradiated embryos. A test was performed to see whether transcriptional activation mediated by p53RE represents a specific response to radiation damage or a common integration point for multiple pathways that lead to excess apoptosis. Beta-galactosidase expression was examined in wild-type and crb embryos carrying either the p53RE-lacZ or the 2kb-lacZ reporter constructs. In stage 12/13 wild-type embryos, expression of the 2kb-lacZ transgene is normally confined to the developing gut but, in similarly aged crb embryos, expression is induced throughout the epidermis. In contrast, the p53RE-lacZ transgene exhibits only basal expression in either wild-type or crb embryos. Thus, despite widespread apoptosis in crb embryos, there is no induction of reporter expression from the p53RE. These results indicate that the p53RE specifically responds to radiation damage, not generally to all proapoptotic signals. They also indicate that irradiation and disrupted development may activate rpr expression through distinct pathways (Brodsky, 2000a).
Drosophila p53 encodes a 385-amino acid protein with significant homology to human p53 in the region of the DNA-binding domain, and to a lesser extent the tetramerization domain. Although Drosophila p53 and human p53 share much sequence and biochemical homology, one major difference between Drosophila p53 and human p53 is that Drosophila p53 lacks the consensus box I sequence found in all vertebrate p53 proteins; this is located in the p53-MDM2 interaction region. Moreover, genome-wide searches in Drosophila have failed to identify an MDM2 homolog. Therefore, MDM2-mediated p53 degradation could be a later evolutionary event. Interestingly, there is a putative PEST region at the N terminus of Drosophila p53 but not human p53. These are P- (proline), E- (glutamate), S- (serine), and T- (threonine) rich sequences flanked by K (lysine) or R (arginine) but not interrupted by any basic amino acids (30), that act as protein degradation signals. It seems possible that Drosophila p53 protein stability is regulated through this PEST sequence instead of the more specific MDM2-p53 autoregulating loop in vertebrates. Purified Drosophila p53 DNA-binding domain protein has been shown to bind to the consensus human p53-binding site by gel mobility analysis. In transient transfection assays, expression of Drosophila p53 in Schneider cells transcriptionally activates promoters that contain consensus human p53-responsive elements. Moreover, a mutant Drosophila p53 (Arg-155 to His-155), like its human p53 counterpart mutant, exerts a dominant-negative effect on transactivation. Ectopic expression of Drosophila p53 in Drosophila eye disc causes cell death and leads to a rough eye phenotype. Drosophila p53 is expressed throughout the development of Drosophila with highest expression levels in early embryogenesis; this high level has a maternal component. Consistent with this, Drosophila p53 RNA levels were high in the nurse cells of the ovary. It appears that p53 is structurally and functionally conserved from flies to mammals. Drosophila will provide a useful genetic system to the further study of the p53 network (Jin, 2000).
The similarity between predicted structure of Drosophila p53 and the crystal structure of the human p53 DNA-binding domain prompted an exploration of whether Drosophila p53 is able to bind to the human p53 consensus binding site. Previous studies have shown that the DNA-binding domain plus N terminus and the DNA-binding domain alone of human p53 have similar affinities for the consensus DNA site as does the full-length protein. Therefore, the fragments of human p53 and Drosophila p53 containing the DNA-binding domains were purified to test their DNA-binding ability. The consensus site for human p53 is PuPuPuCA/T-T/AGPyPyPy-N0-13-PuPuPuCA/T-T/AGPyPyPy. A double-stranded oligonucleotide matching the consensus sequence (5'-TACAGAACATGTCTAAGCATGCTGGG-3') was end labeled and used for gel mobility-shift assay. Drosophila p53 forms a DNA-protein complex as does human p53. The specificity of the DNA-protein interaction was demonstrated by competition assays, in which the unlabeled specific oligonucleotide (SP, the consensus oligonucleotide itself) effectively competes with the labeled probe. The specificity of interaction was demonstrated by using a mutated p53 consensus oligonucleotide (5'-TACAGAAaATtTCTAAGaATtCTGGG-3'; mutation in consensus sequence shown in lowercase) as a probe in a similar gel shift assay. Both Drosophila p53 and human p53 proteins fail to form a complex with the mutated oligonucleotide. The gel mobility-shift analysis demonstrates that the binding affinity of Drosophila p53 to the oligonucleotide sequence is lower than that of human p53. It is not clear whether this was due to the specific oligonucleotide sequence chosen or that Drosophila p53 prefers a similar but a slightly different consensus site than that of human p53 (Jin, 2000).
MAPK phosphatases (MKPs) are important negative regulators of MAPKs in vivo, but ascertaining the role of specific MKPs is hindered by functional redundancy in vertebrates. MKP function was characterized by examining the function of Puckered (Puc), the sole Drosophila Jun N-terminal kinase (JNK)-specific MKP, during embryonic and imaginal disc development. Puc is a key anti-apoptotic factor that prevents apoptosis in epithelial cells by restraining basal JNK signaling. Furthermore, JNK signaling plays an important role in gamma-irradiation-induced apoptosis, and this study examined how JNK signaling fits into the circuitry regulating this process. Radiation upregulates both JNK activity and puc expression in a p53-dependent manner; apoptosis induced by loss of Puc can be suppressed by p53 inactivation. JNK signaling acts upstream of both Reaper and effector caspases. JNK signaling directs normal developmentally regulated apoptotic events. However, if cell death is prevented, JNK activation can trigger tissue overgrowth. Thus, MKPs are key regulators of the delicate balance between proliferation, differentiation and apoptosis during development (McEwen, 2005).
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. 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. Chk2 is a protein kinase that acts downstream of the ataxia telangiectasia mutated (ATM) kinase and may induce cell cycle arrest. 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. Chk1, an evolutionarily conserved protein kinase, implicated in cell cycle checkpoint control in lower eukaryotes, also has been suggested to play a role in p53 regulation. Chk1 also can phosphorylate p53, probably at the same sites as Chk2. 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), Chk1 (grapes), and Chk2 (Loki) 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. 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. 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. 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 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. 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. It has been 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, Chk2 null flies 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).
Genetic and microarray analyses have been used to determine how ionizing radiation (IR) induces p53-dependent transcription and apoptosis in Drosophila melanogaster. IR induces MNK/Chk2-dependent phosphorylation of p53 without changing p53 protein levels, indicating that p53 activity can be regulated without an Mdm2-like activity. In a genome-wide analysis of IR-induced transcription in wild-type and mutant embryos, all IR-induced increases in transcript levels required both p53 and the Drosophila Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper, sickle, and the tumor necrosis factor family member EIGER. Overexpression of Eiger is sufficient to induce apoptosis, but mutations in Eiger do not block IR-induced apoptosis. Animals heterozygous for deletions that span the reaper, sickle, and hid genes exhibited reduced IR-dependent apoptosis, indicating that this gene complex is haploinsufficient for induction of apoptosis. Among the genes in this region, hid plays a central, dosage-sensitive role in IR-induced apoptosis. p53 and MNK/Chk2 also regulate DNA repair genes, including two components of the nonhomologous end-joining repair pathway, Ku70 and Ku80. These results indicate that MNK/Chk2-dependent modification of Drosophila p53 activates a global transcriptional response to DNA damage that induces error-prone DNA repair as well as intrinsic and extrinsic apoptosis pathways (Brodsky, 2004).
The cellular antioxidant defense systems neutralize the cytotoxic by-products referred to as reactive oxygen species (ROS). Among them, selenoproteins have important antioxidant and detoxification functions. The interference in selenoprotein biosynthesis results in accumulation of ROS and consequently in a toxic intracellular environment. The resulting ROS imbalance can trigger apoptosis to eliminate the deleterious cells. In Drosophila, a null mutation in the selD gene (homologous to the human selenophosphate synthetase type 1) causes an impairment of selenoprotein biosynthesis, a ROS burst and lethality. This mutation (known as selDptuf) can serve as a tool to understand the link between ROS accumulation and cell death. To this aim, the mechanism by which selDptuf mutant cells become apoptotic was analyzed in Drosophila imaginal discs. The apoptotic effect of selDptuf does not require the activity of the Ras/MAPK-dependent proapoptotic gene hid, but results in stabilization of the tumor suppressor protein p53 and transcription of the Drosophila pro-apoptotic gene reaper (rpr). Genetic evidence supports the idea that the initiator caspase DRONC is activated and that the effector caspase DRICE is processed to commit selDptuf mutant cells to death. Moreover, the ectopic expression of the inhibitor of apoptosis DIAP1 rescues the cellular viability of selDptuf mutant cells. These observations indicate that selDptuf ROS-induced apoptosis in Drosophila is mainly driven by the caspase-dependent p53/Rpr pathway (Morey, 2003).
Yeast one- and two-hybrid assays were used to examine Dmp53 biochemical functions. For each assay, five Dmp53 derivatives were tested: full-length, i.e., Dmp53; N-terminal fragment, Dmp53(Nt); central DNA-binding fragment, Dmp53(Db); C-terminal fragment, Dmp53(Ct); and full-length with a point mutation in the DNA-binding domain, Dmp53(259H). The 259H 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. To assay for DNA binding, the activation domain of GAL4 was fused to Dmp53 derivatives and they were tested in directed one-hybrid assays. Two reporter constructs were tested for each GAL4-Dmp53 hybrid: the negative control plasmid (pLacZi) contains a minimal promoter upstream of the lacZ gene; the tester plasmid (p53BLUE) contains three copies of a 20 bp consensus binding site for human p53 upstream of the minimal promoter. Full-length Dmp53 is able to activate transcription from the reporter containing human p53 binding sites, but not from the negative control reporter. None of the individual domains (Nt, Db, Ct) were able to activate transcription; significantly, the 259H mutation specifically eliminates activation. These results indicate that Dmp53 can interact with a consensus binding site for human p53 and that a residue required for sequence specific binding in human p53 plays a similar role in Dmp53 (Brodsky 2000a).
To test for transcriptional activation, the DNA-binding domain of GAL4 was fused to Dmp53 derivatives. Three GAL4-dependent reporter constructs were present: the lacZ gene under control of the GAL7 promoter, the ADE2 gene under control of the GAL2 promoter, and the HIS3 gene under control of the GAL1 promoter. While full-length Dmp53 is unable to mediate detectable transcriptional activation of any reporter, Dmp53(Nt) confers modest transcriptional activation of the lacZ reporter construct. However, this derivative does not activate sufficient transcription from the ADE2 and HIS3 constructs to allow growth on plates without adenine and histidine. The weak transcriptional activation due to the N terminus does not provide a strong conclusion about its in vivo function (Brodsky 2000a).
To test for oligomerization, a two-hybrid assay was used with the same reporters as described for the activation assay. Dmp53(Nt) was not tested with the lacZ reporter because it gives a weak positive signal in the activation assay; all other Dmp53 derivatives were tested against themselves in the two-hybrid assay since, tested alone, these fusions are unable to activate the GAL4-dependent reporters. For all three reporters, oligomerization activity is strongest with the Dmp53(Ct) fusions. Full-length Dmp53 and Dmp53(259H) exhibits weaker oligomerization activity in this assay. These results suggest that, despite the lack of sequence similarity in the putative tetramerization domain, the C-terminal region of Dmp53 does contain sequences that can mediate oligomerization (Brodsky 2000a).
The ability of wild-type and mutant forms of Dmp53 to activate transcription in Drosophila S2 cells was tested. Two variants with point mutations in the DNA-binding domain were also used. Dmp53(259H) contains a point mutation that directly disrupts DNA binding. Dmp53(155H) contains a mutation equivalent to the hotspot mutation 175H in human p53; this residue does not directly contact DNA and the mutation may disrupt p53 function by partially unfolding the DNA-binding domain. For each transfection, the localization of Dmp53 was examined using polyclonal antibodies raised against the C terminus of the protein. Wild-type Dmp53 and the two point mutant derivatives localize to the nucleus of transfected cells. In contrast, Dmp53(Ct) is found in both the cytoplasm and the nucleus (Brodsky 2000a).
Two reporter constructs that originally established the transcriptional activity of human p53 were used. One construct, PG13-CAT, contains a multimer of a human p53 binding site upstream of a chloramphenicol acetyl transferase (CAT) reporter gene. A second construct, MG15-CAT, contains a multimer of a mutated site that is not bound by human p53. Transfection of these reporters into S2 cells does not cause increased CAT activity relative to control cells. Cotransfection of PG13-CAT and a vector expressing Dmp53 results in a 10-fold increase in CAT activity. Cotransfection of MG15-CAT and Dmp53 results in only a 2-fold increase in CAT activity. Expression of either point mutant or the C-terminal fragment does not increase activation of the PG13-CAT reporter over background levels. These results demonstrate that wild-type, but not mutant, Dmp53 can activate transcription from a promoter containing binding sites for human p53. Thus, the sequence conservation between human p53 and Dmp53 reflects functional conservation of DNA binding and transcriptional activation (Brodsky 2000a).
Most p53 mutants in human tumors can act as dominant-negative forms, typically leaving the tetramerization domain intact but disrupting DNA binding. Such variants are thought to suppress activity of the wild-type protein through the formation of inactive complexes. A test was performed to see whether the transcriptionally inactive forms of Dmp53 could inhibit transcription mediated by wild-type Dmp53 in S2 cells. Cotransfection of a 3-fold excess of Dmp53(155H) reduces transcription by wild-type Dmp53 by roughly 50% whereas cotransfection of either Dmp53(259H) or Dmp53(Ct) in similar amounts reduces transcription by wild-type Dmp53 to near background levels. Therefore, like their human counterparts, these Dmp53 variants can act as dominant-negative forms that partially or completely block activity of the wild-type protein. The dominant-negative activity of Dmp53(Ct) is consistent with the observation in yeast assays that this domain contains an oligomerization domain (Brodsky 2000a).
To determine whether the sequence similarity of Dmp53 and human p53 may reflect a conserved function as a DNA binding transcription factor, a test was performed to see whether Dmp53 can bind to a double-stranded DNA molecule containing a p53 recognition site using an electrophoretic mobility shift assay. Dmp53 binds specifically to oligonucleotides containing p53 binding sites from the human p21 and GADD45 genes, demonstrating that both DNA binding and target site specificity have been conserved through more than 500 million years of evolution. This interaction is specific, since addition of unlabelled wild-type GADD45 oligoduplex DNA competes for Dmp53 binding, whereas unlabelled mutant GADD45 oligoduplex DNA does not. Moreover, an anti-Dmp53 polyclonal antibody prevents DNA binding by Dmp53, and an anti-Dmp53 monoclonal antibody supershifts the Dmp53/DNA complex. It is interesting that human p53, which was expressed and tested in an identical assay, binds p53 binding sites only in the presence of the activating antibody PAb421. PAb421 is thought to act by associating with a region in the carboxyl terminus of p53 that normally negatively regulates DNA binding. The ability of Dmp53 to bind DNA without any activating treatments may indicate that a similar negative regulatory element does not exist in Dmp53 (Ollmann, 2000).
Dmp53 was expressed in Drosophila larval eye discs using glass-responsive enhancer elements. The glass-Dmp53 (gl-Dmp53) transgene expresses Dmp53 in all cells posterior to the morphogenetic furrow. The morphogenetic furrow marks the front of a wave of cellular differentiation that sweeps from the posterior to the anterior of the eye disc during larval development. Thus, gl-Dmp53 larvae express Dmp53 in all eye disc cells as they differentiate as well as in a subset of cells behind the furrow that undergo a final round of cell division before terminal differentiation. Expression of Dmp53 from the gl-Dmp53 transgene produces viable adults that have small, rough eyes with fused ommatidia. TUNEL staining of gl-Dmp53 eye discs shows that this phenotype is due, at least in part, to widespread apoptosis in cells expressing Dmp53. Similar results are seen when apoptotic cells are detected by acridine orange or Nile Blue. TUNEL-positive cells appear within 15-30 cell diameters of the furrow. Given that the furrow is estimated to move approximately five cell diameters per hour, this indicates that cells initiate apoptosis within 3-6 hr after Dmp53 is expressed (Ollmann, 2000).
The ability of p53 to induce apoptosis in some vertebrate cell types can be inhibited by overexpression of p21. The precise mechanism(s) through which p21 inhibits apoptosis is unknown, but direct inhibitory interactions with procaspase 3 and apoptosis signal-regulating kinase 1 have been reported. To determine if expression of human p21 can similarly suppress Dmp53-induced apoptosis, Dmp53 and p21 were co-expressed in the developing eye disc. p21 expression dramatically suppresses Dmp53-induced apoptosis in the disc as well as the adult rough-eye phenotype. This suppression does not appear to involve reduction of p53 protein levels, since matched disc samples from larvae expressing gl-Dmp53 or gl-Dmp53 plus gl-p21 show similar levels of anti-Dmp53 antibody staining. These data suggest that p53-related proteins in flies and vertebrates trigger apoptosis through similar p21-suppressible pathways. Surprisingly, similar inhibition of apoptosis could not be achieved by coexpression of the baculovirus p35 protein, a universal substrate inhibitor of caspases. Given that p35 inhibits human p53-induced apoptosis in lepidopteran and Drosophila cells, the lack of p35 suppression of apoptosis may reflect different rates and/or levels of Dmp53 and p35 protein accumulation (Ollmann, 2000).
In addition to its ability to affect cell death pathways, mammalian p53 can induce cell cycle arrest at the G1 and G2/M checkpoints. In the Drosophila eye disc, the second mitotic wave is a synchronous, final wave of cell division posterior to the morphogenetic furrow. This unique aspect of development provides a means to assay for similar effects of Dmp53 on the cell cycle. Transition of these cells from G1 to S phase in wild-type discs can be detected by bromodeoxyuridine (BrdU) incorporation into DNA. This transition from G1 to S phase is not blocked or delayed by Dmp53 overexpression from the gl-Dmp53 transgene. In contrast, expression of human p21 or a Drosophila p21 homolog, Dacapo, under control of the same Glass-responsive enhancer element completely blocks DNA replication in the second mitotic wave. However, overexpression of Dmp53 does affect M phase in the eye disc. In wild-type discs, an M phase-specific anti-phospho-histone antibody typically stains a distinct band of cells within the second mitotic wave. In gl-Dmp53 larval eye discs, this band of cells is present but is significantly broader and more diffuse, suggesting that Dmp53 alters the entry into and/or duration of M phase (Ollmann, 2000).
Examination was made of whether loss of Dmp53 function affected apoptosis or cell cycle arrest in response to DNA damage. In order to examine the phenotype of tissues deficient in Dmp53 function, dominant-negative Dmp53 alleles were expressed as transgenes under the control of tissue-specific promoters. Coexpression of Dmp53R155H with wild-type Dmp53 suppresses the rough eye phenotype that normally results from wild-type Dmp53 overexpression, confirming that this mutant protein has dominant-negative activity in vivo. The same result was obtained by expressing the Dmp53H159N protein. Unlike wild-type Dmp53, overexpression of the dominant-negative alleles using the glass enhancer or a constitutive enhancer (arm-GAL4) has no visible effect on normal development (Ollmann, 2000).
In mammalian systems, p53-induced apoptosis plays a crucial role in preventing the propagation of damaged DNA. DNA damage also leads to apoptosis in Drosophila. To determine if this response requires the action of Dmp53, dominant-negative Dmp53 transgenes were overexpressed in the posterior compartment of the wing disc. Wild-type wing discs show widespread apoptosis detectable by TUNEL staining 4 hr after X irradiation. When either dominant-negative allele of Dmp53 is expressed in the posterior compartment of the wing disc, apoptosis is blocked in the cells expressing Dmp53, whereas the anterior compartment displays a normal amount of X ray-induced cell death. Thus, induction of apoptosis following X irradiation requires the function of Dmp53. This proapoptotic role for Dmp53 appears to be limited to a specific response to cellular damage, because developmentally programmed cell death in the eye and other tissues is unaffected by expression of either dominant-negative Dmp53 allele (Ollmann, 2000).
Although the data strongly suggest that Dmp53 function is required for X ray-induced apoptosis, it does not appear to be necessary for the cell cycle arrest induced by the same dose of irradiation. In the absence of irradiation, a random pattern of mitosis is observed in third instar wing discs of Drosophila. Upon irradiation, a cell cycle block in wild-type discs leads to a significant decrease in anti-phospho-histone staining. This cell cycle block is unaffected by expression of dominant-negative Dmp53 in the posterior of the wing disc. Several time points after X irradiation were examined, and all gave similar results, suggesting that both the onset and maintenance of the X ray-induced cell cycle arrest is independent of Dmp53 (Ollmann, 2000).
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