BIOLOGICAL OVERVIEW

Following completion of meiosis, DNA replication must be repressed until fertilization. In Drosophila, this replication block requires the products of the pan gu (png), plutonium (plu) and giant nuclei (gnu) genes. (For information on the Pan Gu legend, see Pan Gu Creates the World). These genes also ensure that S phase oscillates with mitosis in the early division cycles of the embryo. png encodes a Ser/Thr protein kinase expressed only in ovaries and early embryos; the predicted extent of kinase activity in png mutants inversely correlates with the severity of the mutant phenotypes. The Plu and Png proteins form a complex that has Png-dependent kinase activity, and this activity is necessary for normal levels of mitotic cyclins. Cyclin B is a key Png target. Mutations in cyclin B dominantly enhance png, whereas png is suppressed by cyclin B overexpression. Suppression occurs via restoration of Cyclin B protein levels that are decreased in png mutants. plu and gnu phenotypes are also suppressed by cyclin B overexpression. These studies demonstrate that a crucial function of PNG in controlling the cell cycle is to permit the accumulation of adequate levels of Cyclin B protein. These results reveal a novel protein kinase complex that controls S phase at the onset of development apparently by stabilizing mitotic cyclins (Fenger, 2000 and Lee, 2001).

In all species, fertilization of the egg by the sperm triggers two crucial cell cycle changes: an arrest point is relieved and the cell cycle restarts with DNA replication. The cell cycle stage at which the mature egg arrests while awaiting fertilization varies among different species: it can be the G2 phase preceding meiosis, metaphase I or metaphase II of meiosis, or arrest after the completion of meiosis. In Drosophila, the egg is activated to complete meiosis as it passes through the uterus, and fertilization is required for the onset of DNA replication and the cell cycle. Despite the range of possible arrest points, in all organisms fertilization must lead to re-entry into S phase and mitotic division (Fenger, 2000).

The understanding of the molecular changes triggered by fertilization and the potential cell cycle targets of fertilization is still rudimentary. In a number of marine invertebrates and vertebrates, fertilization causes a flux of Ca²⁺ in the egg. This appears to result from activation of phospholipase Cgamma in the egg, leading to an increase in PI3 levels and the cytoplasmic release of intracellular Ca²⁺ pools. It is not known whether such a Ca²⁺ flux also occurs at fertilization in Drosophila or whether phospholipase Cgamma is activated in response to fertilization. It is also not known which cell cycle regulators are affected by fertilization. The control of meiotic arrest and its release by fertilization are best understood in Xenopus, in which cytostatic factor (CSF), an activity dependent on the Mos kinase, maintains the egg in metaphase II by stabilizing high maturation promoting factor (MPF: Cdc2/cyclin B) activity. Fertilization inactivates CSF, although the molecular details of how Ca²⁺ causes this are not known, MPF activity drops and meiosis is completed (Fenger, 2000 and references therein).

Other organisms must also possess regulators that repress cell cycle progression prior to fertilization. As suggested by the fact that Mos has not been found in invertebrates, these regulators may not be universal among organisms. In addition to regulators that may repress the cell cycle only in the mature egg, some organisms may require specific cell cycle regulators during the early embryonic divisions. In organisms with rapid embryonic development such as marine invertebrates, amphibians and insects, the early embryonic divisions occur via a simplified cell cycle in which S phase alternates with mitosis. In these embryonic cycles, growth and transcription do not occur, and the S-M oscillations are driven by maternal components stockpiled into the egg during oogenesis. The post-transcriptional control of these early cell cycles might be through unique regulators of DNA replication and nuclear division. More likely, unique molecules might impose altered control of conserved cell cycle regulators that also function during the archetypal G1-S-G2-M cell cycle (Fenger, 2000).

During the first seven cell cycles in Drosophila, the mitotic cyclins A and B, and CDC2 activity are present at high levels with no detectable fluctuation in overall levels during the cell cycle. Localized degradation of Cyclin B has been detected, though, and it is thought that localization of cyclins A and B may be important in coordinating the nuclear cycles with the microtubules. Furthermore, inhibition of the cyclin degradation machinery with a destruction box peptide or injection of non-destructible Cyclin B causes mitotic arrest in the early embryo. All these results suggest that cyclins play a crucial role in coordinating S phase and mitosis in the early embryo. How the cyclins are controlled is unclear (Fenger, 2000).

In Drosophila there are three maternal effect genes, plutonium, pan gu and giant nuclei, that are needed both to inhibit DNA replication in the unfertilized egg and to control the S-M cycles of embryogenesis. Females mutant for any one of these genes produce eggs that complete meiosis but do not arrest, in contrast to unfertilized eggs from wild-type females. Instead, all four meiotic products inappropriately undergo DNA replication to become polyploid, often fusing into a single nucleus as they increase in ploidy. If the eggs from plu, png or gnu mutant mothers are fertilized, they undergo defective S-M cycles: DNA replication takes place, but nuclear division does not occur, resulting in giant polyploid nuclei similar to the mutant unfertilized eggs. Intriguingly, the centrosomes dissociate from the nuclei and continue to replicate and divide in mutant embryos, indicating that nuclear regulation has been uncoupled from the centrosome cycle (Fenger, 2000).

There appears to be an active requirement for the function of these genes during the S-M cycles, because weak alleles of png permit transient S-M cycling before nuclear division ceases (Shamanski, 1991). These weak mutations yield embryos with up to 16 giant polyploid nuclei. The three genes control the same biological process, because mutations in plu or gnu dominantly enhance the phenotype of weak png mutations, eliminating the transient S-M cycling (Shamanski, 1991). Thus the phenotypic analysis of mutations in plu, png and gnu indicates that these genes are necessary to inhibit S phase prior to fertilization, and they are needed to coordinate S and M during early embryogenesis (Fenger, 2000).

The plu gene has been shown to encode a 19 kDa ankyrin-repeat protein (Axton, 1994). png encodes a Ser/Thr protein kinase that exists in vivo in a complex with Plu. This protein kinase complex is present specifically in the egg and early embryo, and thus it appears to control S phase uniquely in response to fertilization at the onset of development. Analysis of cyclins A and B in png mutants shows that Png affects the levels and post-translational modification of mitotic cyclins, as well as the extent of histone H3 phosphorylation and CDC2 kinase activity. Therefore Png controls chromosome condensation and mitotic progression in the early embryo (Fenger, 2000).

The phenotypes of plu, png and gnu mutants suggest that these genes normally function to inhibit initiation of DNA replication during early embryogenesis. Activating mitosis is one means by which this could be accomplished. If mitotic functions were not activated in the mutant embryos then repeated rounds of DNA replication could occur. This phenomenon would be similar to the repeated rounds of DNA replication that result from loss-of-function of cdc2 and cyclin B in S. pombe. Mitotic cyclin/Cdk activity is needed to inactivate replication origins, and chromosome condensation in mitosis may also serve to block DNA replication. To test if png, plu and gnu affect regulation of mitosis, the protein levels and forms of CDC2 and the mitotic cyclins A and B were examined in mutant embryos. Interestingly, the levels of mitotic cyclins are decreased in mutant embryonic extracts, and the effect is allele specific with respect to png. Three forms of Cyclin A were detected on immunoblots, and the fastest migrating form was predominantly decreased in the mutants: barely detectable levels of this fast migrating form were present in weak png mutant embryos, and none was detected in strong png, plu or gnu mutants. The slower two forms of Cyclin A were less affected, although they also showed allele-specific reduction: the greatest decrease was seen in png¹⁰⁵⁸, png¹⁷², png¹⁹²⁰ , plu and gnu, and the highest levels of Cyclin A were seen in the three weak png alleles. This experiment was repeated twice, and the same decreases in Cyclin A levels were observed. It was also found that levels of Cyclin A were reduced in unfertilized eggs from png¹⁰⁵⁸ mutant mothers (Fenger, 2000).

To determine if the missing fast-migrating Cyclin A form was phosphorylated, and thus a potential Png substrate, wild-type embryonic extracts were treated with lambda-phosphatase. Compared with the 'no phosphatase' control and to wild-type extracts homogenized directly in urea sample buffer, phosphatase-treated extracts showed a loss of the slowest migrating form and an accumulation of the fastest migrating form, indicating that the fast form was actually unphosphorylated. If Cyclin A were a substrate of Png, the phosphorylated form of Cyclin A would be the most decreased of the forms. Since the unphosphorylated form appeared to be preferentially lost in png mutants, it is unlikely that Cyclin A is a Png substrate (Fenger, 2000).

An allele-dependent decrease in Cyclin B levels of mutant embryos was also detected. Again, all of the alleles of png, gnu and the plu null mutation showed decreased cyclin levels compared with wild-type embryos, and of the mutants, the highest levels were seen in the three weak png alleles. The plu null and gnu mutants had the lowest levels, and of the png alleles, the lowest levels were seen in png¹⁷², png¹⁹²⁰, png¹⁵⁸ and png¹⁰⁵⁸. It is interesting that png¹⁷² and png¹⁹²⁰ (which were both predicted to have the lowest kinase activity based on the nature of the mutations) also have the lowest levels of cyclins A and B. Cyclin B levels also were decreased in unfertilized eggs from png¹⁰⁵⁸ mutant mothers. Consistent with the decrease in levels of mitotic cyclins, it was found that CDC2 kinase activity was also decreased (Fenger, 2000).

Examination of the levels and forms of CDC2 showed no difference between wild-type and mutant embryos. It has been observed that Histone H3 phosphorylation correlates with CDC2 kinase activity in early Drosophila embryos, so anti-phospho-H3 antibodies were used to examine levels of H3 phosphorylation in mutant embryos. Phospho-H3 levels were decreased in all the mutants, and again the degree of decrease correlated with allele strength. The lowest levels of phospho-H3 were seen in plu, gnu, png¹⁷², png¹⁹²⁰ and png¹⁰⁵⁸. The highest levels were observed in the three weak png alleles and png¹⁵⁸. MPM2 antibodies also recognize phospho-epitopes present in mitotic cells and dependent on CDC2 activity. A decrease in at least one MPM2 epitope was observed in png¹⁰⁵⁸ and plu⁶ embryonic extracts compared with wild. To assay CDC2 kinase levels directly, CDC2 was immunoprecipitated and levels of histone H1 kinase activity were tested in the pellets. The proteins in the pellet were immunoblotted and the levels of phosphorylated histone H1 were compared with the amount of CDC2 immunoprecipitated, as measured by probing the membrane with antibodies against CDC2. In the mutants the levels of CDC2 kinase activity were decreased about twofold. In conclusion, these results show that mitotic cyclins and CDC2 activity are decreased in png, plu and gnu mutants, and the decrease is allele specific and consistent with predicted levels of Png kinase activity (Fenger, 2000).

There may be a requirement for an S phase inhibitor in the mature egg because the cytoplasm is stockpiled with maternal replication components: the meiotic products may be especially susceptible to initiation of DNA replication when the chromosomes decondense and go through an interphase-like state after completing meiosis. It is also possible that a homolog or functional analog is expressed zygotically, and therefore expression of Png is not required during later development. The phenotype of png mutants suggests that the Png kinase pathway must be regulated temporally and spatially: temporally, in order to inhibit replication during M phase but not S phase, and spatially, to inhibit S phase in the polar bodies, but not the zygotic nuclei. This regulation may be at the level of Png localization and activity, although it has not been possible to localize Plu and Png within the embryo by antibody staining. Alternatively, Plu and Png activity may be upstream of spatially and temporally controlled cell cycle regulators (Fenger, 2000).

As a new protein kinase, orthologs of Png have not yet been identified in other species. It seems likely that other organisms in which the embryonic divisions proceed via a rapid S-M cycle may contain orthologs of Png that control these early cycles. It is also possible that early embryonic cell cycle regulators are more divergent than other cell cycle regulators, because the coordination of meiosis, fertilization, and the first mitosis is divergent in different species. Although not a sequence homolog, in many ways the Png kinase parallels Mos, a Ser/Thr kinase unique to vertebrates. Similar to Png, Mos inhibits DNA replication until fertilization by promoting MPF activity, perhaps primarily by stabilizing mitotic cyclins. In contrast to Png, however, Mos is also required for entrance into meiosis and inhibition of DNA replication between the meiotic divisions, and it is degraded during the first mitosis (Fenger, 2000 and references therein).

The Plu protein controls the same cell cycle processes as Png, and biochemical observations support the idea that the two proteins act together. Plu and Png are present in a complex in mature eggs and embryos. Furthermore, functional Png is needed for it to be complexed with Plu. This may be because Png and Plu interact through an adapter protein that is a target of Png. Png and Plu did not interact in the yeast two-hybrid system, supporting this hypothesis. The levels of Plu protein are depressed in strong png mutants (Elfring, 1997), and this appears to result from a modified, unstable form of Plu present in png mutants. The functional relationship between Plu and Png remains to be determined. One possibility is that Plu is a downstream effector of Png activity, but it is also possible that Plu activates the Png kinase (Fenger, 2000).

The primary defect in png, plu and gnu mutants is likely caused by a decrease in the levels of mitotic cyclins A and B and associated CDK activity. The over-replication phenotype might result because mitotic cyclins are required to block re-replication in the unfertilized egg and early embryo, because the embryo requires chromosome condensation to block re-replication, or a combination of both. Work in Schizosaccharomyces pombe has shown that Cyclin B can function to inhibit DNA replication, since strains deleted for cdc13, which encodes Cyclin B, undergo repeated rounds of S phase without mitoses. Moreover, it has been shown that CDC2 and Cyclin A inhibit endoreplication in diploid cells during Drosophila larval development. The replication of centrosomes that are unattached to nuclei, seen in these mutants, is observed also in embryos lacking Cyclin B, so this mutant cytoskeletal defect may be due to decreased Cyclin B levels (Fenger, 2000).

It is unlikely that Png interacts directly with cyclins because Png and Plu do not co-immunoprecipitate with Cyclin A or Cyclin B, and the unphosphorylated form of Cyclin A appears to be predominantly lost in png mutants. The differential decrease of the different forms of Cyclin A suggests that it is unstable in the mutants, particularly the faster migrating form, and that the decrease is not due to decreased cyclin translation. One possibility is that Png controls cyclin stability by acting through the protein degradation machinery of the APC/cyclosome. It is known that yeast Cdh1, an activator of the APC and subsequent Clb degradation, is inactivated by phosphorylation, so Fizzy-related (Fzr), the Drosophila Cdh1 homolog, could be a substrate of Png. The identification of the Plu-Png protein kinase complex opens the way to identify its regulators and substrate targets in controlling the S-M cell cycles. In a survey of the genome to identify genes that genetically interact to suppress or enhance png mutations, several intervals and loci have been identified. Recovery of Png interactors will permit the unraveling of the mechanism by which Png stabilizes mitotic cyclins and inhibits S phase at this time in development (Fenger, 2000).

GENE STRUCTURE

cDNA clone length - 1126

Bases in 5' UTR - 80

Exons - 1

Bases in 3' UTR - 170

PROTEIN STRUCTURE

Amino Acids - 291

Structural Domains

To determine the biochemical function of the png gene product, the png gene was cloned by positional cloning. Complementation tests between png mutations and deficiencies, and duplications delineate png to a 130 kb region. Using genomic DNA clones from the region in germline transformation rescue experiments, a 1.7 kb fragment capable of rescuing the maternal effect lethality of png minus homozygotes was isolated. Sequencing of the fragment reveals an open reading frame (ORF) that spans most of the 1.7 kb and is contained entirely within it, suggesting that it is the png gene. To identify the png transcript, Drosophila ovary cDNA clones were isolated that hybridized to the rescuing fragment. The longest cDNA is 1126 bp, not including the poly-A tail, and the 5' end of the transcript starts 80 bp upstream of the ATG. The sequence 5' to the ATG contains an in frame stop codon and no other ATG codons in any frame. Alignment of the 1.7 kb genomic sequence with the eight cDNAs shows no introns, indicating that the png transcript is a single exon. Conceptual translation of the longest cDNA reveals that the Png protein has strong homology to Ser/Thr kinases. The protein shows highest homology to members of the Snf1/AMP kinase family, with 27% amino acid identity to Snf1 over 265 amino acids. Phylogenetic analysis, however, shows that Png is more distantly related to Snf1 family members than they are to each other, and probably represents a new family of Ser/Thr kinases. png is small, encoding a 291 amino acid protein with a predicted molecular weight of 33 kDa, that contains only a catalytic domain. The absence of a regulatory domain suggests that separately encoded regulatory subunits may associate with Png (Fenger, 2000).

date revised: 28 November 2001

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