Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in theXenopusegg

Abstract

SummaryFertilization triggers release from meiotic arrest and initiates events that prepare 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 phospho-regulation is still unknown. We examined absolute protein and phospho-site dynamics after fertilization by mass spectrometry-based proteomics. To do this, we developed a new approach for calculating the stoichiometry of phospho-sites from multiplexed proteomics that is compatible with dynamic, stable and multi-site 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 as well as new components of the block to polyspermy. The analytical approaches demonstrated here are broadly applicable to studies of dynamic biological systems.

Selective dephosphorylation by PP2A-B55 directs the meiosis I-meiosis II transition in oocytes

“…We also note that this limited protein turnover during meiosis is consistent with prior reports in other organisms (Kishimoto, 2018b;Peuchen et al, 2017;Presler et al, 2017). However, despite this broad stability of protein levels, there were several notable exceptions (Figure 1F).…”

“…We undertook a proteomic and phosphoproteomic strategy using oocytes of the sea star Patiria miniata, which undergo meiosis with high synchrony (Swartz et al, 2019). Prior analyses have revealed proteome-wide changes in animal models including Xenopus, Drosophila, and sea urchins (Guo et al, 2015;Krauchunas et al, 2012;Presler et al, 2017;Zhang et al, 2019). However, the biology of these organisms limits access to a comprehensive series of time points spanning prophase I through the embryonic divisions, including the critical meiosis I/II transition.…”

Phosphoproteome dynamics during mitotic exit in budding yeast

“…In the future, it will be important to complement this with measurements of absolute phosphosite occupancies. Methods to do so have been developed (Presler et al , ), but limit the number of sites that can be interrogated. Kinase motif signatures are easily discernible, which allowed us to portray the impact of kinases on phosphoproteome dynamics.…”

A calcium‐mediated actin redistribution at egg activation in Drosophila

“…This calcium rise, which can result from influx of extracellular calcium and/or the release of calcium from internal stores, leads to dramatic changes in the mature oocyte that prepare it for further development. Many of these changes require the activity of calcium‐dependent kinases and phosphatases (Horner et al, ; Mochida & Hunt, ; Takeo, Hawley, & Aigaki, ; Takeo, Tsuda, Akahori, Matsuo, & Aigaki, ), which alter the phosphoproteome of the egg (Guo et al, ; Krauchunas, Horner, & Wolfner, ; Presler et al, ; Roux, Radeke, Goel, Mushegian, & Foltz, ; Zhang, Ahmed‐Braimah, Goldberg, & Wolfner, ; reviewed in Krauchunas & Wolfner, ).…”