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

Open AccessPublished:November 26, 2018DOI:https://doi.org/10.1074/mcp.RA118.001076
      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.

      Graphical Abstract

      Egg activation encompasses a series of major changes through which a resting mature oocyte transitions to an active state that is capable of cell proliferation and embryo development. During this process, the oocyte's vitelline membrane undergoes biochemical modifications, the meiotic cell cycle is released from its species-specific arrest (metaphase II in vertebrates; metaphase I in Drosophila), and the oocyte transcriptome and proteome are remodeled through post-transcriptional and post-translational regulation (
      • Horner V.L.
      • Wolfner M.F.
      Transitioning from egg to embryo: triggers and mechanisms of egg activation.
      ,
      • Krauchunas A.R.
      • Wolfner M.F.
      Molecular changes during egg activation.
      ,
      • Machaty Z.
      • Miller A.R.
      • Zhang L.
      Egg activation at fertilization.
      ,
      • Marcello M.R.
      • Singson A.
      Fertilization and the oocyte-to-embryo transition in C. elegans.
      ,
      • Laver J.D.
      • Marsolais A.J.
      • Smibert C.A.
      • Lipshitz H.D.
      Regulation and function of maternal gene products during the maternal-to-zygotic transition in Drosophila.
      ,
      • Von Stetina J.R.
      • Orr-Weaver T.L.
      Developmental control of oocyte maturation and egg activation in metazoan models.
      ,
      • Aviles-Pagan E.E.
      • Orr-Weaver T.L.
      Activating embryonic development in Drosophila.
      ). These intricately regulated events transform the oocyte from a highly differentiated gamete into a totipotent zygote.
      In vertebrates and many invertebrate species, fertilization triggers egg activation (
      • Knott J.G.
      • Kurokawa M.
      • Fissore R.A.
      • Schultz R.M.
      • Williams C.J.
      Transgenic RNA interference reveals role for mouse sperm phospholipase C zeta in triggering Ca2+ oscillations during fertilization.
      ,
      • Saunders C.M.
      • Larman M.G.
      • Parrington J.
      • Cox L.J.
      • Royse J.
      • Blayney L.M.
      • Swann K.
      • Lai F.A.
      PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development.
      ,
      • Steinhardt R.
      • Zucker R.
      • Schatten G.
      Intracellular calcium release at fertilization in the sea urchin egg.
      ,
      • Yoon S.Y.
      • Eum J.H.
      • Lee J.E.
      • Lee H.C.
      • Kim Y.S.
      • Han J.E.
      • Won H.J.
      • Park S.H.
      • Shim S.H.
      • Lee W.S.
      • Fissore R.A.
      • Lee D.R.
      • Yoon T.K.
      Recombinant human phospholipase C zeta 1 induces intracellular calcium oscillations and oocyte activation in mouse and human oocytes.
      ). But in insects, the trigger is independent of fertilization and is, instead, passage through the female reproductive tract (
      • Horner V.L.
      • Wolfner M.F.
      Mechanical stimulation by osmotic and hydrostatic pressure activates Drosophila, oocytes in vitro in a calcium-dependent manner.
      ,
      • Kaneuchi T.
      • Sartain C.V.
      • Takeo S.
      • Horner V.L.
      • Buehner N.A.
      • Aigaki T.
      • Wolfner M.F.
      Calcium waves occur as Drosophila, oocytes activate.
      ,
      • Sartain C.V.
      • Wolfner M.F.
      Calcium and egg activation in Drosophila.
      ,
      • Went D.F.
      • Krause G.
      Alteration of egg architecture and egg activation in an endoparasitic Hymenopteran as a result of natural or imitated oviposition.
      ,
      • York-Andersen A.H.
      • Parton R.M.
      • Bi C.J.
      • Bromley C.L.
      • Davis I.
      • Weil T.T.
      A single and rapid calcium wave at egg activation in Drosophila.
      ). Both types of trigger cause a rise in Ca2+ level in the oocyte cytoplasm, which is thought to set off the subsequent events by activating Ca2+/calmodulin-dependent kinase II (CaMKII)
      The abbreviations used are: CamKII, Ca2+/Calmodulin dependent kinase II; ACN, acetonitrile; APC/C, anaphase promoting complex/cyclosome; CanA, calcineurin A; CanB2, calcineurin B2; CDK, cyclin-dependent kinase; CID, collision induced dissociation; FA, formic Acid; HCD-MS/MS, higher energy collisional dissociation-MS/MS; hpRP, high pH reverse phase; kd, knockdown; MSA, multistage activation; NL, neutral loss trigger; PP2A-B55, protein phosphatase 2A at B55; Pp2B-14D, protein phosphatase 2B at 14D; SPS, synchronous precursor selection; TMT, tandem mass tag.
      1The abbreviations used are: CamKII, Ca2+/Calmodulin dependent kinase II; ACN, acetonitrile; APC/C, anaphase promoting complex/cyclosome; CanA, calcineurin A; CanB2, calcineurin B2; CDK, cyclin-dependent kinase; CID, collision induced dissociation; FA, formic Acid; HCD-MS/MS, higher energy collisional dissociation-MS/MS; hpRP, high pH reverse phase; kd, knockdown; MSA, multistage activation; NL, neutral loss trigger; PP2A-B55, protein phosphatase 2A at B55; Pp2B-14D, protein phosphatase 2B at 14D; SPS, synchronous precursor selection; TMT, tandem mass tag.
      and the phosphatase calcineurin (CN) (
      • Sartain C.V.
      • Wolfner M.F.
      Calcium and egg activation in Drosophila.
      ,
      • Sanders J.R.
      • Swann K.
      Molecular triggers of egg activation at fertilization in mammals.
      ). These enzymes are presumed to transduce the Ca2+ signal downstream by phosphorylating or dephosphorylating maternally deposited proteins.
      Consistent with this scheme, large-scale protein phosphorylation state changes during egg activation have been detected in proteomic studies of vertebrates and invertebrates (
      • Guo H.
      • Garcia-Vedrenne A.E.
      • Isserlin R.
      • Lugowski A.
      • Morada A.
      • Sun A.
      • Miao Y.
      • Kuzmanov U.
      • Wan C.
      • Ma H.
      • Foltz K.
      • Emili A.
      Phosphoproteomic network analysis in the sea urchin Strongylocentrotus purpuratus, reveals new candidates in egg activation.
      ,
      • Roux M.M.
      • Townley I.K.
      • Raisch M.
      • Reade A.
      • Bradham C.
      • Humphreys G.
      • Gunaratne H.J.
      • Killian C.E.
      • Moy G.
      • Su Y.H.
      • Ettensohn C.A.
      • Wilt F.
      • Vacquier V.D.
      • Burke R.D.
      • Wessel G.
      • Foltz K.R.
      A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation.
      ,
      • Krauchunas A.R.
      • Horner V.L.
      • Wolfner M.F.
      Protein phosphorylation changes reveal new candidates in the regulation of egg activation and early embryogenesis in D. melanogaster.
      ,
      • Presler M.
      • Van Itallie E.
      • Klein A.M.
      • Kunz R.
      • Coughlin M.L.
      • Peshkin L.
      • Gygi S.P.
      • Wühr M.
      • Kirschner M.W.
      Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in the Xenopus, egg.
      ). In Drosophila, more than 300 proteins are subject to phosphoregulation during egg activation (
      • Krauchunas A.R.
      • Horner V.L.
      • Wolfner M.F.
      Protein phosphorylation changes reveal new candidates in the regulation of egg activation and early embryogenesis in D. melanogaster.
      ), and orthologs of more than 55% of these proteins are also phosphoregulated during sea urchin egg activation (
      • Guo H.
      • Garcia-Vedrenne A.E.
      • Isserlin R.
      • Lugowski A.
      • Morada A.
      • Sun A.
      • Miao Y.
      • Kuzmanov U.
      • Wan C.
      • Ma H.
      • Foltz K.
      • Emili A.
      Phosphoproteomic network analysis in the sea urchin Strongylocentrotus purpuratus, reveals new candidates in egg activation.
      ). These deeply conserved phosphoproteomic changes suggest their functional importance during this transition, and our genetic data show that these phosphoregulated proteins include numerous factors required for oogenesis and embryogenesis (
      • Zhang Z.
      • Krauchunas A.R.
      • Huang S.
      • Wolfner. M.F.
      Maternal proteins that are phosphoregulated upon egg activation include crucial factors for oogenesis, egg activation and embryogenesis in Drosophila, melanogaster.
      ). These results support the idea that phosphoregulation is an important mechanism that remodels the oocyte proteome to prepare the cell to undertake development.
      Here, we focus on the role of calcineurin in egg activation. This enzyme acts as a heterodimer consisting of a catalytic subunit A and a calcium-sensing regulatory subunit B (
      • Rusnak F.
      • Mertz P.
      Calcineurin: form and function.
      ). The Drosophila genome encodes 3 variants of calcineurin subunit A (PP2B-14D, CanA-14F and CanA1) and 2 variants of calcineurin subunit B (CanB and CanB2). Among these variants, only PP2B-14D, CanA-14F, and CanB2 are significantly expressed in the ovary (flyatlas.org). Oocytes depleted of the calcineurin regulatory subunit CanB2 are able to briefly resume meiosis from metaphase I arrest upon egg activation, but eventually they arrest again in anaphase I (
      • Takeo S.
      • Hawley R.S.
      • Aigaki T.
      Calcineurin and its regulation by Sra/RCAN is required for completion of meiosis in Drosophila.
      ). On the other hand, constitutive activation of the calcineurin catalytic subunit PP2B14D (CnAact) causes a range of meiotic defects in mature oocytes (
      • Takeo S.
      • Hawley R.S.
      • Aigaki T.
      Calcineurin and its regulation by Sra/RCAN is required for completion of meiosis in Drosophila.
      ). These results indicate that both the function and proper regulation of calcineurin are required for meiosis progression in the activating eggs of Drosophila. Consistent with this model, calcineurin in Xenopus, is transiently activated following the first Ca2+ influx, and it promotes Metaphase II exit by removing inhibitory phosphorylations from the APC/C component Apc3 and its co-activator Fzy (
      • Nishiyama T.
      • Yoshizaki N.
      • Kishimoto T.
      • Ohsumi K.
      Transient activation of calcineurin is essential to initiate embryonic development in Xenopus laevis.
      ,
      • Mochida S.
      • Hunt T.
      Calcineurin is required to release Xenopus egg extracts from meiotic M phase.
      ).
      Calcineurin is also involved in other aspects of egg activation in Drosophila. Depletion of CanB2 inhibits the dephosphorylation and activation of the Gnu and YA proteins (
      • Krauchunas A.R.
      • Sackton K.L.
      • Wolfner M.F.
      Phospho-regulation pathways during egg activation in Drosophila melanogaster.
      ). Gnu is a regulatory subunit of Pan Gu kinase (
      • Freeman M.
      • Glover D.M.
      The gnu mutation of Drosophila, causes inappropriate DNA synthesis in unfertilized and fertilized eggs.
      ,
      • Renault A.D.
      • Zhang X.H.
      • Alphey L.S.
      • Frenz L.M.
      • Glover D.M.
      • Saunders R.D.
      • Axton J.M.
      Giant nuclei is essential in the cell cycle transition from meiosis to mitosis.
      ), a crucial regulator of maternal mRNA translation and cell cycle progression in the early embryo (
      • Kronja I.
      • Yuan B.
      • Eichhorn S.W.
      • Dzeyk K.
      • Krijgsveld J.
      • Bartel D.P.
      • Orr-Weaver T.L.
      Widespread changes in the posttranscriptional landscape at the Drosophila, oocyte-to-embryo transition.
      ), and YA is a maternal protein essential for the initiation of embryonic mitosis (
      • Lin H.F.
      • Wolfner M.F.
      The Drosophila, maternal-effect gene fs(1)Ya, encodes a cell cycle-dependent nuclear envelope component required for embryonic mitosis.
      ,
      • Liu J.
      • Song K.
      • Wolfner M.F.
      Mutational analyses of fs(1)Ya, an essential, developmentally regulated, nuclear envelope protein in Drosophila.
      ,
      • Lopez J.M.
      • Song K.
      • Hirshfeld A.B.
      • Lin H.
      • Wolfner M.F.
      The Drosophila, fs(1)Ya protein, which is needed for the first mitotic division, is in the nuclear lamina and in the envelopes of cleavage nuclei, pronuclei, and nonmitotic nuclei.
      ). In addition, the calcineurin regulator Sarah (Sra; calcipressin) is also required at the transition from oocyte to embryo (
      • Takeo S.
      • Hawley R.S.
      • Aigaki T.
      Calcineurin and its regulation by Sra/RCAN is required for completion of meiosis in Drosophila.
      ,
      • Takeo S.
      • Tsuda M.
      • Akahori S.
      • Matsuo T.
      • Aigaki T.
      The calcineurin regulator sra plays an essential role in female meiosis in Drosophila.
      ,
      • Horner V.L.
      • Czank A.
      • Jang J.K.
      • Singh N.
      • Williams B.C.
      • Puro J.
      • Kubli E.
      • Hanes S.D.
      • McKim K.S.
      • Wolfner M.F.
      • Goldberg M.L.
      The Drosophila, calcipressin sarah is required for several aspects of egg activation.
      ). Depletion of Sra leads to anaphase I arrest in activated eggs and prevents other events including polyadenylation of crucial maternal transcripts and maturation of the male pronucleus (
      • Takeo S.
      • Tsuda M.
      • Akahori S.
      • Matsuo T.
      • Aigaki T.
      The calcineurin regulator sra plays an essential role in female meiosis in Drosophila.
      ,
      • Horner V.L.
      • Czank A.
      • Jang J.K.
      • Singh N.
      • Williams B.C.
      • Puro J.
      • Kubli E.
      • Hanes S.D.
      • McKim K.S.
      • Wolfner M.F.
      • Goldberg M.L.
      The Drosophila, calcipressin sarah is required for several aspects of egg activation.
      ). These findings together suggest that calcineurin is at the center of the regulatory mechanisms that drive the transition from oocyte to embryo in Drosophila.
      Here, we analyze the proteomic and phosphoproteomic changes that take place during egg activation and characterize how these changes are affected by the depletion or constitutive activation of calcineurin in female germ cells. Our data reveal that calcineurin activity is crucial for regulating hundreds of phosphosites during this developmental transition. The proteins whose phosphorylation states are influenced by calcineurin include important regulators of egg activation events, particularly meiotic cell cycle progression and protein translation. Calcineurin-regulated proteins are also enriched for functional groups related to biological processes that are crucial later in development during early embryogenesis. Our results reveal the scale of calcineurin's impact on the remodeling of the oocyte proteome during egg activation.

      DISCUSSION

      Phosphoregulation of maternal proteins has been suggested as a major mechanism that drives the transition from oocyte to embryo (
      • Guo H.
      • Garcia-Vedrenne A.E.
      • Isserlin R.
      • Lugowski A.
      • Morada A.
      • Sun A.
      • Miao Y.
      • Kuzmanov U.
      • Wan C.
      • Ma H.
      • Foltz K.
      • Emili A.
      Phosphoproteomic network analysis in the sea urchin Strongylocentrotus purpuratus, reveals new candidates in egg activation.
      ,
      • Roux M.M.
      • Townley I.K.
      • Raisch M.
      • Reade A.
      • Bradham C.
      • Humphreys G.
      • Gunaratne H.J.
      • Killian C.E.
      • Moy G.
      • Su Y.H.
      • Ettensohn C.A.
      • Wilt F.
      • Vacquier V.D.
      • Burke R.D.
      • Wessel G.
      • Foltz K.R.
      A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation.
      ,
      • Krauchunas A.R.
      • Horner V.L.
      • Wolfner M.F.
      Protein phosphorylation changes reveal new candidates in the regulation of egg activation and early embryogenesis in D. melanogaster.
      ,
      • Presler M.
      • Van Itallie E.
      • Klein A.M.
      • Kunz R.
      • Coughlin M.L.
      • Peshkin L.
      • Gygi S.P.
      • Wühr M.
      • Kirschner M.W.
      Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in the Xenopus, egg.
      ). Therefore, the activities and control of protein phosphoregulators are of great importance to understanding egg activation and early embryo development. Calcineurin is a highly conserved phosphoregulator that responds to a Ca2+ rise, the first common step to egg activation pathways in various organisms. Several previous studies showed that calcineurin plays a central role in regulating pathways that mediate egg activation events in Drosophila (
      • Krauchunas A.R.
      • Wolfner M.F.
      Molecular changes during egg activation.
      ,
      • Takeo S.
      • Hawley R.S.
      • Aigaki T.
      Calcineurin and its regulation by Sra/RCAN is required for completion of meiosis in Drosophila.
      ,
      • Takeo S.
      • Tsuda M.
      • Akahori S.
      • Matsuo T.
      • Aigaki T.
      The calcineurin regulator sra plays an essential role in female meiosis in Drosophila.
      ,
      • Horner V.L.
      • Czank A.
      • Jang J.K.
      • Singh N.
      • Williams B.C.
      • Puro J.
      • Kubli E.
      • Hanes S.D.
      • McKim K.S.
      • Wolfner M.F.
      • Goldberg M.L.
      The Drosophila, calcipressin sarah is required for several aspects of egg activation.
      ), but the molecular consequences of calcineurin activity at the oocyte-to-egg transition were unknown.
      Here, we utilized quantitative proteomic methods to gain a more comprehensive view of calcineurin's influence on global and phospho-proteomes during egg activation. Our data reveal widespread misregulation of protein phosphorylation changes in CanB2 depleted eggs. In particular, the majority of phosphosites whose abundances change during control egg activation are misregulated when CanB2 is depleted, pointing to calcineurin as an upstream master regulator of multiple egg activation events. Calcineurin may dephosphorylate certain of these sites directly, but other cases, particularly those in which the lack of calcineurin is correlated with decreases in the phosphosite's abundance, must be mediated indirectly through other phosphatases and kinases. An additional potential twist is that phosphosites within the calcineurin catalytic subunits PP2B-14D and CanA-14F are misregulated in CanB2 kd, oocytes. One of these sites, found in both proteins, matches the Cdk1 consensus recognition site (
      • Songyang Z.
      • Blechner S.
      • Hoagland N.
      • Hoekstra M.F.
      • Piwnica-Worms H.
      • Cantley L.C.
      Use of an oriented peptide library to determine the optimal substrates of protein kinases.
      ,
      • Heifetz Y.
      • Yu J.
      • Wolfner M.F.
      Ovulation triggers activation of Drosophila, oocytes.
      ) and is conserved in human calcineurin. Calcineurin autoregulation thus might impart yet another level of regulation to the pathway.

      Calcineurin Plays an Unanticipated Role during Oocyte Maturation

      Although CanB2-deficient mature oocytes do not show any obvious morphological defects and are arrested in what appears to be a cytologically normal metaphase I state, these oocytes display clear abnormalities in their phosphoproteomes. Almost all of the 50 misregulated phosphopeptides identified are subject to downregulations (in the sense that their abundances were lower in CanB2 kd, than in control oocytes). Conversely, mature oocytes produced by females expressing a constitutively active form of calcineurin (CnAact) also misregulate several phosphopeptides, but here most of these abnormalities are upregulations. These results together imply that calcineurin plays heretofore unexpected active roles in oocyte maturation that must be mediated by other enzymes. Our results that Akap200 is mis-phospho-regulated in CanB2 kd, oocytes suggest Akap200 as a candidate for mediating at least some of calcineurin's regulatory effects in oocytes.
      Because the calcineurin's activity usually is dependent on intracellular calcium, it will be important to investigate the mechanism by which calcineurin regulates these phosphorylation sites before the calcium wave initiating egg activation. It will also be of great interest to explore whether and how these misregulated phosphorylations are important for the functions of the target proteins in the context of the developing oocyte.

      What Is the Molecular/Cellular State of the Arrested CanB2 kd Eggs?

      Our images of the activated eggs produced by CanB2 kd, mothers indicate a consistent arrest in what appears to be anaphase of meiosis I, with homologous chromosomes that have separated from each other supplemental Fig. S1C,) (
      • Takeo S.
      • Hawley R.S.
      • Aigaki T.
      Calcineurin and its regulation by Sra/RCAN is required for completion of meiosis in Drosophila.
      ). These activated eggs differ from mature oocytes from the CanB2 kd, female, which appear to be in metaphase of meiosis I because all chromosomes are congressed at the metaphase plate (
      • Takeo S.
      • Hawley R.S.
      • Aigaki T.
      Calcineurin and its regulation by Sra/RCAN is required for completion of meiosis in Drosophila.
      ). Passage through the female's reproductive tract, the trigger for egg activation in insects [12–16, 81], thus causes a change in at least some aspects of the cell cycle status of CanB2 kd, eggs.
      Our results indicate however that at the molecular level, activated CanB2 kd, eggs still largely resemble metaphase I (Fig. 8): The phosphoproteome and global proteomes of these calcineurin-deficient eggs are little changed by the activation process. Cdk1 activity remains high; in fact, the Cdk activity may be even higher than prior to activation because the levels of the T14 and Y15 inhibitory phosphorylations on Cdk1 decrease. Activation does not decrease the phosphorylation of Gwl kinase and Endosulfine in the mutant eggs, indicating that the Cdk-countering phosphatase PP2A-B55 remains inactive. In these eggs, the APC/C does not signal the destruction of Matrimony protein as normally occurs during egg activation.
      Figure thumbnail gr8
      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.
      These observations explain why activated CanB2 kd, eggs become arrested in a cell cycle state not very far removed from that in mature oocytes. However, our results do not explain the most remarkable feature of this arrest: that homologous chromosomes apparently separate, based on the images of the CanB2 kd, embryos (supplemental Fig. S1C,) (
      • Takeo S.
      • Hawley R.S.
      • Aigaki T.
      Calcineurin and its regulation by Sra/RCAN is required for completion of meiosis in Drosophila.
      ). The basis for this separation in Drosophila meiosis is somewhat mysterious because the genome has no obvious homologs of the meiotic α-kleisin Rec8. The destruction of Securin and the resultant activation of Separase are still needed in flies to release arm cohesion in anaphase I, even though the target of Separase remains unclear (
      • Guo Z.
      • Batiha O.
      • Bourouh M.
      • Fifield E.
      • Swan A.
      Role of Securin, Separase and Cohesins in female meiosis and polar body formation in Drosophila.
      ). One possibility for how arm cohesion could be partially released independently of calcineurin involves the fact that egg activation may also turn on the calcium-dependent kinase CamKII. This enzyme might potentiate a pathway leading to the destruction of a small population of Securin and thus some degree of homologous chromosome separation. An alternative model involves misregulation of a mechanism mediated by the Wapl protein that can remove cohesin independently of Separase (
      • Eichinger C.S.
      • Kurze A.
      • Oliveira R.A.
      • Nasmyth K.
      Disengaging the Smc3/kleisin interface releases cohesin from Drosophila, chromosomes during interphase and mitosis.
      ) and is known to influence meiotic chromosome segregation in flies (
      • Verni F.
      • Gandhi R.
      • Goldberg M.L.
      • Gatti M.
      Genetic and molecular analysis of wings apart-like (wapl), a gene controlling heterochromatin organization in Drosophila melanogaster.
      ). This second pathway is normally thought to occur in prophase, but we know of no data that would preclude its function during the metaphase arrest in mature oocytes, particularly in those from CanB2 kd, mothers. Unfortunately, our data do not allow us to discriminate among these or other possible models to explain the separation of homologous chromosomes during the activation of mutant eggs. No known component of cohesin complexes or any other candidate molecules known to be involved in either of these pathways showed significant changes in abundance in control or CanB2 kd, eggs relative to the mature oocytes, so further biochemical studies will be required to solve this puzzle.
      Another unexplained observation is the unexpected behavior of two key phosphosites: the T14 and Y15 inhibitory phosphorylations on Cdk1. T14 and Y15 are highly conserved target sites of Wee1, a kinase that is downregulated by Cdks; and of Cdc25, a phosphatase that is up-regulated by Cdks (
      • Den Haese G.J.
      • Walworth N.
      • Carr A.M.
      • Gould K.L.
      The Wee1 protein kinase regulates T14 phosphorylation of fission yeast Cdc2.
      ,
      • Watanabe N.
      • Broome M.
      • Hunter T.
      Regulation of the human WEE1Hu CDK tyrosine 15-kinase during the cell cycle.
      ,
      • Berry L.D.
      • Gould K.L.
      Regulation of Cdc2 activity by phosphorylation at T14/Y15.
      ,
      • Hoffmann I.
      • Clarke P.R.
      • Marcote M.J.
      • Karsenti E.
      • Draetta G.
      Phosphorylation and activation of human cdc25-C by cdc2–cyclin B and its involvement in the self-amplification of MPF at mitosis.
      ,
      • Kumagai A.
      • Dunphy W.G.
      Regulation of the cdc25 protein during the cell cycle in Xenopus, extracts.
      ). One would thus predict that in wildtype, the phospho-occupancy of these sites would be low in mature oocytes (M phase) but high after egg activation (interphase). Yet the phosphorylation of these sites was lower in control (and CanB2 kd,) eggs compared with oocytes (supplemental Table S1). It must be noted that we do not know the absolute occupancy level of these sites, so it is possible that during these developmental stages, the majority of Cdk1 molecules are never phosphorylated at these sites. But whatever the absolute occupancy, T14 and Y15 becomes dephosphorylated in some Cdk1 molecules upon egg activation. At least in wildtype, this behavior can perhaps be rationalized by the status of the polar bodies. In Drosophila, polar bodies generated during meiosis are not expelled from the egg but are instead sequestered at the periphery of the egg and fuse together to form the polar body rosette (
      • Mahowald A.P.
      • Goralski T.J.
      • Caulton J.H.
      In vitro activation of Drosophila, eggs.
      ). Because the rosette is maintained in an arrested state with condensed chromosomes in early embryos (
      • Su T.T.
      • Sprenger F.
      • DiGregorio P.J.
      • Campbell S.D.
      • O'Farrell P.H.
      Exit from mitosis in Drosophila syncytial embryos requires proteolysis and cyclin degradation, and is associated with localized dephosphorylation.
      ), it is possible that the dephosphorylation of Cdk1 T14 and Y15 in control activated eggs reflects the formation and maintenance of this structure.

      Calcineurin Helps Determine the Phosphorylation State of Several Translation Regulators

      We have verified the previously reported role of calcineurin on Gnu dephosphorylation, leading to the activation of PNG) (
      • Lee L.A.
      • Van Hoewyk D.
      • Orr-Weaver T.L.
      The Drosophila cell cycle kinase PAN GU forms an active complex with PLUTONIUM and GNU to regulate embryonic divisions.
      ,
      • Hara M.
      • Petrova B.
      • Orr-Weaver T.L.
      Control of PNG kinase, a key regulator of mRNA translation, is coupled to meiosis completion at egg activation.
      ). Indeed, we found that several proteins whose translation is known to be turned on by PNG were up-regulated during activation of control eggs but not during the activation of eggs deficient in calcineurin. Our results strongly suggest that calcineurin also influences the phosphorylation state of several translation regulators in addition to Gnu (Fig. 6C,) (Fig. 8). These regulatory proteins (as well as Gnu) may be direct substrates of calcineurin because all of these sites become dephosphorylated when Ca2+ levels increase during control egg activation. Further investigation is necessary in order to explore the functional importance of the phosphorylation state of these translation factors.

      GSK3β and Calcineurin May Act Within a Regulatory Loop

      We were interested to observe that a key inhibitory phosphosite on GSK3β (Ser9) both is up-regulated upon egg activation and is dependent on calcineurin activity. Our data are consistent with, but do not prove, the idea that calcineurin's effect on this phosphosite is indirect and mediated by the kinases Akt1 and possibly TORC2. In turn, GSK3β has been shown to regulate Sra (
      • Takeo S.
      • Swanson S.K.
      • Nandanan K.
      • Nakai Y.
      • Aigaki T.
      • Washburn M.P.
      • Florens L.
      • Hawley R.S.
      Shaggy/glycogen synthase kinase 3beta and phosphorylation of Sarah/regulator of calcineurin are essential for completion of Drosophila, female meiosis.
      ), a protein that inhibits calcineurin in mature eggs but that acts as a phosphorylation-dependent calcineurin activator during egg activation. These facts suggest the model that egg activation involves a negative feedback loop that helps create transiency in calcineurin activity during egg activation (Fig. 8). Calcineurin activity initiated by the Ca2+ rise leads indirectly to increased phosphorylation of GSK3β-Ser9, decreased Sra phosphorylation, and thus decreased calcineurin activity. This auto-shutoff model is consistent with a previous report in Xenopus, that calcineurin activity is transient following the calcium wave, and that both the initial calcineurin activation and its transiency are crucial for transition into embryogenesis (
      • Mochida S.
      • Hunt T.
      Calcineurin is required to release Xenopus egg extracts from meiotic M phase.
      ). Future work will be needed to test this negative feedback model for Drosophila and other organisms during the egg activation process.

      Data Availability

      Mass spectrometry proteomics data have been deposited into the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset accession number PXD010991. The R analysis pipeline is available as a GitHub repository (https://github.com/YazBraimah/Dmel_eggAct_phosphoproteomics).

      Acknowledgments

      We thank Dr. Tim Karr for the opportunity to contribute to this Special Issue, and NIH grant R21-HD088744 to M.F.W. for funding this study. We thank Drs. Terry Orr-Weaver (Massachusetts Institute of Technology) and Toshiro Aigaki for their kind gifts of anti-Gnu primary antibodies and CnAact flies, respectively. We thank Drs. Marcus Smolka (Cornell University), Steve Dorus (Syracuse University), Kathryn Lilley (University of Cambridge), and Sheng Zhang (Cornell BioResource Center) for helpful advice on the relative merits of different quantitative mass spectrometry methods, and Drs. John Schimenti and Ken Kemphues for helpful comments on an early version of this manuscript, and anonymous reviewers for helpful comments on this version. We thank the Proteomic and MS Facility of Cornell University's BioResource Center for running the mass spectrometry on their Orbitrap Fusion mass spectrometer (which had been purchased with NIH grant SIG 1S10 OD017992). We thank the TRiP consortium at Harvard Medical School (funded by NIH grant NIGMS R01-GM084947) and the Bloomington Stock Center for transgenic RNAi fly stocks used in this study.

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