No transcription, no problem: Protein phosphorylation changes and the transition from oocyte to embryo

Abstract

Although mature oocytes are arrested in a differentiated state, they are provisioned with maternally-derived macromolecules that will start embryogenesis. The transition to embryogenesis, called 'egg activation', occurs without new transcription, even though it includes major cell changes like completing stalled meiosis, translating stored mRNAs, cytoskeletal remodeling, and changes to nuclear architecture. In most animals, egg activation is triggered by a rise in free calcium in the egg's cytoplasm, but we are only now beginning to understand how this induces the egg to transition to totipotency and proliferation. Here, we discuss the model that calcium-dependent protein kinases and phosphatases modify the phosphorylation landscape of the maternal proteome to activate the egg. We review recent phosphoproteomic mass spectrometry analyses that revealed broad phospho-regulation during egg activation, both in number of phospho-events and classes of regulated proteins. Our interspecies comparisons of these proteins pinpoints orthologs and protein families that are phospho-regulated in activating eggs, many of which function in hallmark events of egg activation, and others whose regulation and activity warrant further study. Finally, we discuss key phospho-regulating enzymes that may act apically or as intermediates in the phosphorylation cascades during egg activation. Knowing the regulators, targets, and effects of phospho-regulation that cause an egg to initiate embryogenesis is crucial at both fundamental and applied levels for understanding female fertility, embryo development, and cell-state transitions.

Keywords: Calcineurin; Calcium; CamKII; Egg activation; Embryogenesis; Fertilization; Meiotic arrest; Oocyte; Post-translational control; Protein phosphorylation; Translation; Zygote.

Fig. 1. Overview of calcium-mediated phosphorylation events that lead to release from meiotic arrest.

(A) Calcium rise at the start of egg activation stimulates calcium-dependent enzymes (CaMKII and Calcineurin; Calmodulin not depicted). CDK1-Cyclin B (MPF) activity is high, while APC/C activity is off. Emi2 is active and competitively inhibits APC/C subunit APC10 from binding its activator. (A1) CaMKII phosphorylates Emi2 at T195 to create a docking site for PLK1. CaMKII also phosphorylates Wee1B kinase at S15 to activate it. (A2) Calcineurin dephosphorylates Emi2 at S335, which destabilizes its interaction with PP2A. Calcineurin also dephosphorylates Cdc20 at T68, which allows Cdc20 to perform its role as a cofactor for APC/C. (B) Intermediate kinases propagate phospho-signaling started by CaMKII and Calcineurin. (B3) Wee1B phosphorylates CDK1 at its inhibitory Y15 site, promoting CDK1’s disassociation from Cyclin B and deactivation of MPF. (B4) PLK1 recognizes the binding site on Emi2 resulting from CaMKII phosphorylation. PLK1 then phosphorylates Emi2 at its phospho-degron site S32. (C) Emi2 is degraded and inactivated, relieving its inhibition of APC/C; thus, APC/C is activated to release metaphase arrest. (C5) Emi2 phosphorylation at its phospho-degron promotes degradation and subsequent deactivation of Emi2 via the SCF degradation complex. (C6) APC/C is activated by association with its cofactor Cdc20 and activator Ube2S and marks Cyclin B for degradation, along with components of the metaphase arrest machinery like Securin (not shown). Orange glow around a protein represents that its activity is on. # = MAPK-regulated sites. * = CDK1 sites. A-C represent temporal progression of metaphase arrest release, but 1–6 do not imply order of events (e.g. CaMKII and Calcineurin are simultaneously activated by calcium/Calmodulin). Phospho-sites depicted were all identified in Xenopus except for Wee1B S15 (found in mouse). As discussed in Section 4.2, CaMKII and Calcineurin are not both known to be involved in all organisms, but are both needed in Xenopus.

Fig. 2. Calcium rise at egg activation simulates phospho-protein regulation by CaMKII and Calcineurin.

Raised free cytoplasmic calcium (Ca²⁺) activates CaMKII and/or Calcineurin via Calmodulin (CaM). Orange haze indicates active proteins. CaMKII’s known targets during egg activation are Emi2 and Wee1B, as identified in vertebrates (Xenopus and mouse, respectively). Calcineurin regulates (directly or indirectly, hence the dotted line beneath Calcineurin) the phosphorylation states of dozens of maternal proteins. Those shown are the Drosophila proteins whose orthologs were identified in multiple species’ phospho-mass spectrometry screens (bolded proteins in the Drosophila row of Table 2). For technical reasons, CaM activity has not been directly assessed during egg activation; a dotted line therefore connects Ca²⁺ to CaM, and CaM to CaMKII/Calcineurin.