Phosphoproteomic network analysis in the sea urchin Strongylocentrotus purpuratus reveals new candidates in egg activation

Abstract

Fertilization triggers a dynamic symphony of molecular transformations induced by a rapid rise in intracellular calcium. Most prominent are surface alterations, metabolic activation, cytoskeletal reorganization, and cell-cycle reentry. While the activation process appears to be broadly evolutionarily conserved, and protein phosphorylation is known to play a key role, the signaling networks mediating the response to fertilization are not well described. To address this gap, we performed a time course phosphoproteomic analysis of egg activation in the sea urchin Strongylocentrotus purpuratus, a system that offers biochemical tractability coupled with exquisite synchronicity. By coupling large-scale phosphopeptide enrichment with unbiased quantitative MS, we identified striking changes in global phosphoprotein patterns at 2- and 5-min postfertilization as compared to unfertilized eggs. Overall, we mapped 8796 distinct phosphosite modifications on 2833 phosphoproteins, of which 15% were differentially regulated in early egg activation. Activated kinases were identified by phosphosite mapping, while enrichment analyses revealed conserved signaling cascades not previously associated with egg activation. This work represents the most comprehensive study of signaling associated with egg activation to date, suggesting novel mechanisms that can be experimentally tested and providing a valuable resource for the broader research community. All MS data have been deposited in the ProteomeXchange with identifier PXD002239 (http://proteomecentral.proteomexchange.org/dataset/PXD002239).

Maternal Proteins That Are Phosphoregulated upon Egg Activation Include Crucial Factors for Oogenesis, Egg Activation and Embryogenesis inDrosophila melanogaster

“…Consistent with this hypothesis, 174 conserved proteins alter their phosphorylation states during the egg-to-embryo transition in both sea urchin and the Drosophila (Krauchunas et al 2012, Guo et al 2015), suggesting that a cohort of phosphoregulated proteins is deeply conserved, and may be functionally important for the transition from oocyte to embryo.…”

“…Guo et al (2015) observed 174 proteins in common between our dataset of proteins phospho-modulated during Drosophila egg activation (Krauchunas et al 2012) and their set of phosphoregulated proteins during this same transition in sea urchin. This led them to suggest that there are conserved cassettes of protein functions needed for this important developmental transition.…”

“…During egg activation, the oocyte proteome is dynamically regulated through degradation and post-translational modification (Guo et al 2015, Krauchunas et al 2012, Kronja et al 2014a, Laver et al 2015, Presler et al 2017, Roux et al 2006). Among the latter processes, changes in protein phosphorylation state are very prevalent during this transition.…”

“…In Drosophila melanogaster , over 300 maternal proteins alter their phosphorylation state during egg activation (Krauchunas et al 2012), including a few previously shown to be crucial for egg activation and the onset of syncytial divisions, such as Young arrest (YA) and Giant nuclei (GNU) (Lin & Wolfner 1991, Renault et al 2003, Sackton et al 2009, Yu et al 1999). In sea urchins, changes at hundreds of phosphosites were reported to occur within 2 min and 5 min post fertilization (Guo et al 2015, Roux et al 2006), and in Xenopus ∼500 phosphosites show dynamic regulation following fertilization (Presler et al 2017). Consistent with the remodeling of oocyte phosphoproteome seen during the egg-to-embryo transition, two highly-conserved calcium-dependent regulators that are essential for egg activation in several species, Ca 2+ /Calmodulin dependent kinase II (CaMKII) ( Xenopus and mouse (Backs et al 2010, Morin et al 1994)) and calcineurin ( Xenopus and Drosophila; (Horner et al 2006, Mochida & Hunt 2007, Takeo et al 2010, Takeo et al 2006)), are a kinase and a phosphatase respectively (Hudmon & Schulman 2002, Rusnak & Mertz 2000).…”

An unregulated regulator: Vasa expression in the development of somatic cells and in tumorigenesis

“…This fact implies that post-transcriptional modifications of Vasa are essential for its differential activities in each lineage and/or role. Vasa (DDX4) protein also appears phosphorylated in sea urchins (Guo et al, 2015), and potentially in mice and humans based on the search in the PhosphoSite proteomic database (http://www.phosphosite.org; Gustafson and Wessel, 2010), yet no detailed analysis has been provided especially in somatic cells. In the future, accomplishing the phosphor-proteomics analysis in the germ line, embryonic cells, regenerative tissues, and in tumor cells will likely reveal if Vasa undergoes differential modifications for each cellular process and for each development stage.…”