Calcineurin-dependent Protein Phosphorylation Changes During Egg Activation in Drosophila melanogaster

Abstract

In almost all animals studied to date, the crucial process of egg activation, by which an arrested mature oocyte transitions into an actively developing embryo, initiates with an increase in Ca²⁺ in the oocyte's cytoplasm. This Ca²⁺ rise sets off a series of downstream events, including the completion of meiosis and the dynamic remodeling of the oocyte transcriptome and proteome, which prepares the oocyte for embryogenesis. Calcineurin is a highly conserved phosphatase that is activated by Ca²⁺ upon egg activation and that is required for the resumption of meiosis in Xenopus,, ascidians, and Drosophila. The molecular mechanisms by which calcineurin transduces the calcium signal to regulate meiosis and other downstream events are still unclear. In this study, we investigate the regulatory role of calcineurin during egg activation in Drosophila melanogaster,. Using mass spectrometry, we quantify the phosphoproteomic and proteomic changes that occur during egg activation, and we examine how these events are affected when calcineurin function is perturbed in female germ cells. Our results show that calcineurin regulates hundreds of phosphosites and also influences the abundance of numerous proteins during egg activation. We find calcineurin-dependent changes in cell cycle regulators including Fizzy (Fzy), Greatwall (Gwl) and Endosulfine (Endos); in protein translation modulators including PNG, NAT, eIF4G, and eIF4B; and in important components of signaling pathways including GSK3β and Akt1. Our results help elucidate the events that occur during the transition from oocyte to embryo.

Keywords: Drosophila melanogaster; calcineurin; egg activation; phosphorylation; proteomics.

Graphical abstract

Fig. 1.

Schematic of experimental set up. Please see text for details.

Fig. 2.

TMT-based quantitative proteomic analysis captures prevalent protein phosphorylation changes in egg activation. A,, Volcano plots of egg/oocyte phosphopeptide abundance foldchange in control and CanB2 kd, using MSA acquisition method. B,, Volcano plots of egg/oocyte phosphopeptide abundance foldchange in control and CanB2 kd, using NL acquisition method. C,, Comparison of significantly changed phosphopeptides (in control) identified by MSA and NL methods. D,, The abundance changes of proteins that contain significantly changed phosphopeptides.

Fig. 3.

Germline depletion of CanB2 causes widespread misregulation of phosphorylation sites during egg activation. A,, Comparison of egg/oocyte phosphopeptide abundance foldchange in control and CanB2 kd, sample sets as quantified by MSA and NL methods. B,, Comparison of phosphopeptides that significantly increased or decreased during egg activation in control and in CanB2 kd, sample sets, as quantified by MSA and NL methods. C,, Enrichment analysis shows the terms that are significantly enriched among proteins whose phosphorylation states are misregulated during egg activation in CanB2 kd, sample sets (MSA and NL combined). n.s. = non-significant.

Fig. 4.

Perturbation of calcineurin activity in female germline leads to misregulation of protein phosphorylation states in mature oocytes. A,, Volcano plot of CanB2 kd,/control oocyte phosphopeptide abundance ratio as quantified by MSA and NL methods. B,, Terms enriched among proteins whose phosphorylation states are misregulated in CanB2 kd, mature oocytes. C,, Volcano plot of CnA^act,/control oocyte phosphopeptide abundance ratio as quantified by MSA and NL methods. Gene Ontology categories: BP = biological process, CC = cellular compartment, MF = molecular function.

Fig. 5.

Absence of CanB2 leads to misregulation of phosphorylation, as well as protein level of cell cycle regulators during egg activation. Y-axis is the log2 fold change of abundance in eggs compared with that in oocytes. A,, phosphopeptide abundance change of Endos[S68] and Gwl[T606] during egg activation in control and CanB2 kd,. Error bars reflect standard errors. B,, Mtrm and CycB3 protein level change during egg activation in control and CanB2 kd,. Error bars reflect standard errors. C,, Abundance of various Fzy phosphopeptides and Fzy protein level during egg activation in control and CanB2 kd,. Error bars reflect standard errors.

Fig. 6.

Protein translation is disrupted in activated eggs with CanB2 kd,. A,, Gnu phosphosites that are dephosphorylated upon egg activation in wt but not CanB2 kd, activated eggs. Error bars reflect standard errors. B,, Abundance changes of 3 proteins that are known to be translated upon egg activation under the regulation of PNG. Error bars reflect standard errors. C,, Aberrant phosphoregulation was observed at various phosphosites on translation regulators NAT1, eIF4G and eIF4B in CanB2 kd,. Error bars reflect standard errors.

Fig. 7.

Sgg is strongly phosphorylated at the S9 Akt1 inhibitory phosphorylation site during egg activation in control but not in CanB2 kd,. A,, Y-axis is the log2 fold change of abundance in eggs compared with that in oocytes. Error bars reflect standard errors. B,, Western blotting for Akt1 S505 phosphorylation and Akt1 protein (Akt1-pan) in mature oocytes and activated unfertilized eggs produced by control or CanB2 kd, females.

Fig. 8.

Schematic summary of the influences of calcineurin during egg activation on meiotic cell cycle (orange), translation regulation (blue), and GSK3β (green). Dashed lines indicate possible direct or indirect regulatory relationship, solid lines indicate direct regulatory relationship.