Academic journal article Genetics

Bypassing the Greatwall-Endosulfine Pathway: Plasticity of a Pivotal Cell-Cycle Regulatory Module in Drosophila Melanogaster and Caenorhabditis Elegans

Academic journal article Genetics

Bypassing the Greatwall-Endosulfine Pathway: Plasticity of a Pivotal Cell-Cycle Regulatory Module in Drosophila Melanogaster and Caenorhabditis Elegans

Article excerpt

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-phase-specific 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 CDK-directed 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 mammalian tissue culture cells, a different phosphatase, PP2A associated with a B55-type regulatory subunit, is primarily responsible for mitotic phosphosite dephosphorylation (Mochida et al. 2009). PP2A/B55 is inhibited during M phase by a pathway featuring Greatwall kinase (Gwl) and its substrates in the Endosulfine (Endos) family (Castilho et al. 2009; Mochida et al. 2009, 2010; Vigneron et al. 2009; Gharbi-Ayachi et al. 2010). Endos proteins phosphorylated by Gwl bind to PP2A/B55 and inhibit its phosphatase activity in Xenopus extracts (Gharbi-Ayachi et al. 2010; Mochida et al. 2010), and Drosophila melanogaster oocytes lacking Endos have reduced levels of phosphorylated epitopes despite normal Cdk1/cyclinB activity (Von Stetina et al. 2008). Xenopus Gwl is activated at least in part by MPF, ensuring PP2A/B55 inhibition specifically during M phase (Yu et al. 2006; Blake- Hodek et al. 2012). When Gwl or Endosulfine are depleted, frog egg extracts can neither enter nor maintain M phase (Yu et al. 2006; Zhao et al. 2008; Gharbi-Ayachi et al. 2010; Mochida et al. 2010), mammalian tissue culture cells exhibit delays at the G2/M transition (Burgess et al. 2010; Voets and Wolthuis 2010), and Drosophila oocytes fail to progress to metaphase I (Von Stetina et al. 2008).

In this report, we investigate the Gwl-Endos-PP2A/B55 pathway during several types of cell division in two invertebrate organisms, D. …

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