Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in the Xenopus egg

Abstract

Fertilization releases the meiotic arrest and initiates the events that prepare the egg for the ensuing developmental program. Protein degradation and phosphorylation are known to regulate protein activity during this process. However, the full extent of protein loss and phosphoregulation is still unknown. We examined absolute protein and phosphosite dynamics of the fertilization response by mass spectrometry-based proteomics in electroactivated eggs. To do this, we developed an approach for calculating the stoichiometry of phosphosites from multiplexed proteomics that is compatible with dynamic, stable, and multisite phosphorylation. Overall, the data suggest that degradation is limited to a few low-abundance proteins. However, this degradation promotes extensive dephosphorylation that occurs over a wide range of abundances during meiotic exit. We also show that eggs release a large amount of protein into the medium just after fertilization, most likely related to the blocks to polyspermy. Concomitantly, there is a substantial increase in phosphorylation likely tied to calcium-activated kinases. We identify putative degradation targets and components of the slow block to polyspermy. The analytical approaches demonstrated here are broadly applicable to studies of dynamic biological systems.

Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit

“…A simple view would be to separate these functions based on APC/C coactivator, CDC20 for cyclin destruction and CDH1 for resetting cell status. This is supported by a proteomic analysis of meiotic exit in Xenopus eggs which found that wide-spread dephosphorylation was triggered by the degradation of a small number of proteins, typified by the APC/C CDC20 substrates: CCNB1/2, securin and GMNN (Presler et al, 2017). These eggs lack CDH1, therefore typical APC/C CDH1 substrates such as Aurora kinases were, unsurprisingly, stable.…”

“…Global proteomic analysis of meiotic exit in Xenopus eggs, which lack CDH1, showed that proteolysis was limited to a few key rapidly degraded targets such as cyclin B1, B2 and securin and that dephosphorylation proceeded normally in this system (Presler et al, 2017). Additionally, proteasome inhibition, using MG-132, did not alter the rate of dephosphorylation of PPP1CA-pT320 or PRC1-pT481 in HeLa cells following CDK inhibition (Cundell et al, 2013).…”

“…These eggs lack CDH1, therefore typical APC/C CDH1 substrates such as Aurora kinases were, unsurprisingly, stable. The absence of CDH1-dependent degradation is thought to help bypass G1 and promote rapid divisions within the early embryo (Presler et al, 2017), highlighting a role for APC/C CDH1 activity in promoting the formation of a stable G1 state. Nevertheless, APC/C CDC20 is required for the degradation of BUBR1, CENPF and PLK1 late in anaphase (Choi et al, 2009;Floyd et al, 2008;Gurden et al, 2010).…”

Rapid and site-specific deep phosphoproteome profiling by data-independent acquisition (DIA) without the need for spectral libraries

“…A high fractional stoichiometry combined with dynamic regulation is a strong indication that the site is functional in the cellular context studied 1 . It is possible to determine the absolute occupancy or fractional stoichiometry of phosphorylation sites on a large scale by using ratios observed in both the phosphopeptide, its non-phosphorylated counterpart peptide and the respective protein between treatment conditions from SILAC data 22 and TMT-multiplexed data 23,24 .…”

A calcium‐mediated actin redistribution at egg activation in Drosophila

“…further development. Many of these changes require the activity of calcium-dependent kinases and phosphatases (Horner et al, 2006;Mochida & Hunt, 2007;Takeo, Hawley, & Aigaki, 2010;Takeo, Tsuda, Akahori, Matsuo, & Aigaki, 2006), which alter the phosphoproteome of the egg (Guo et al, 2015;Krauchunas, Horner, & Wolfner, 2012;Presler et al, 2017;Roux, Radeke, Goel, Mushegian, & Foltz, 2008;Zhang, Ahmed-Braimah, Goldberg, & Wolfner, 2019;reviewed in Krauchunas & Wolfner, 2013).…”