Bypassing the Greatwall–Endosulfine Pathway: Plasticity of a Pivotal Cell-Cycle Regulatory Module in Drosophila melanogaster and Caenorhabditis elegans

Abstract

In vertebrates, mitotic and meiotic M phase is facilitated by the kinase Greatwall (Gwl), which phosphorylates a conserved sequence in the effector Endosulfine (Endos). Phosphorylated Endos inactivates the phosphatase PP2A/B55 to stabilize M-phasespecific phosphorylations added to many proteins by cyclin-dependent kinases (CDKs). We show here that this module functions essentially identically in Drosophila melanogaster and is necessary for proper mitotic and meiotic cell division in a wide variety of tissues. Despite the importance and evolutionary conservation of this pathway between insects and vertebrates, it can be bypassed in at least two situations. First, heterozygosity for loss-of-function mutations of twins, which encodes the Drosophila B55 protein, suppresses the effects of endos or gwl mutations. Several types of cell division occur normally in twins heterozygotes in the complete absence of Endos or the near absence of Gwl. Second, this module is nonessential in the nematode Caenorhaditis elegans. The worm genome does not contain an obvious ortholog of gwl, although it encodes a single Endos protein with a surprisingly well-conserved Gwl target site. Deletion of this site from worm Endos has no obvious effects on cell divisions involved in viability or reproduction under normal laboratory conditions. In contrast to these situations, removal of one copy of twins does not completely bypass the requirement for endos or gwl for Drosophila female fertility, although reducing twins dosage reverses the meiotic maturation defects of hypomorphic gwl mutants. These results have interesting implications for the function and evolution of the mechanisms modulating removal of CDKdirected phosphorylations. ENTRY into M phase of the cell cycle is driven by the rapid activation of the kinase MPF (M phase promoting factor; Cdk1/cyclin B), which then phosphorylates hundreds of target proteins at proline-directed phosphosites (S/T P) (Dephoure et al. 2008;Lindqvist et al. 2009). These sites must remain phosphorylated during M phase, but then be dephosphorylated upon M-phase exit. It would make sense that the phosphatase(s) directed against mitotic phosphosites should be inhibited during M phase, because if not, these key phosphorylations would be prematurely removed, preventing cells from entering M phase.Recent findings support this hypothesis. During M phase in Saccharomyces cerevisiae, the phosphatase Cdc14p, whose activity against MPF targets is required for mitotic exit, is sequestered in the nucleolus from its substrates (reviewed in Mocciaro and Schiebel 2010). In Xenopus egg extracts or in

Quantitative proteomics reveals the dynamics of protein changes during Drosophila oocyte maturation and the oocyte-to-embryo transition

“…The timing of maturation likely is controlled by keeping Cyclin B/CDK1 activity at bay through inhibition of Polo kinase by Matrimony (Mtrm) and downstream activation of the PP2A phosphatase inhibitor Endos (19)(20)(21). After a still elusive signal initiates maturation, an oocyte-specific cytoplasmic poly(A) polymerase, the product of the wispy gene, leads to extension of poly(A) tails (22,23).…”

Role for regulated phosphatase activity in generating mitotic oscillations in Xenopus cell-free extracts

“…3 C and D). This simulation explains why Greatwall and ENSA are dispensable during mitotic cycles of Drosophila embryos heterozygotic for PP2A:B55δ (39) and in Caenorhabditis elegans (40). This finding also explains why previous cell-cycle models [e.g., Ciliberto's (24)] with constitutive Cdk1 counteracting phosphatases were permissive for oscillations.…”

Cell Cycle Regulators in Female Meiosis of Drosophila melanogaster

“…The ability of Endos to bind and inhibit PP2A appears to in turn be regulated by the phosphorylation of Endos by Greatwall kinase (Gwl) [16,17]. Gwl was initially discovered in Drosophila by the dominant Scant allele, and was found to play multiple roles in meiosis beyond NEB [18].…”

“…Gwl was initially discovered in Drosophila by the dominant Scant allele, and was found to play multiple roles in meiosis beyond NEB [18]. Both genetic and biochemical evidence points to a simple linear pathway in which Gwl phosphorylation of Endos allows Endos to bind and inhibit PP2A [16,17,19,20]. While Gwl has not yet been directly implicated in oocyte maturation, transgenic Endos with a S68A mutation, abolishing the Gwl phosphorylation site, is unable to rescue the delayed NEB phenotype of a Endos null mutant [17].…”

“…Both genetic and biochemical evidence points to a simple linear pathway in which Gwl phosphorylation of Endos allows Endos to bind and inhibit PP2A [16,17,19,20]. While Gwl has not yet been directly implicated in oocyte maturation, transgenic Endos with a S68A mutation, abolishing the Gwl phosphorylation site, is unable to rescue the delayed NEB phenotype of a Endos null mutant [17]. Thus Gwl-mediated phosphorylation likely plays a role in the Endos-mediated inhibition of PP2A to promote NEB (Figure 1).…”