Identification of Proteins Interacting with Fyn Kinase at Fertilization in Patiria miniata

Abstract

Release of Ca2+ from the endoplasmic reticulum is the integral molecular mechanism responsible for initiating development at fertilization. The components of the signaling pathway controlling this Ca2+ release are not fully known in any organism. Eggs and sperm of the starfish, Patiria miniata, present an attractive model to study the molecular aspects of fertilization because of the ability to obtain millions of viable sperm and eggs, along with the ease of in vitro fertilization. Two Src family kinases (SFKs) and the lipase PLCγ have been shown to be involved in this signaling pathway and are necessary for Ca2+ release. However, additional interacting proteins and the receptor(s) initiating this pathway have not been identified or characterized. In this study, GST fusion protein constructs of the SH2 domains of Fyn (a SFK) were prepared and affinity interactions were performed at different time points before and after sperm addition to eggs. The affinity complexes were subjected to Western blotting to visualize the interacting proteins. Using an anti‐phosphotyrosine antibody, 5 distinct bands indicate that tyrosine phosphorylated egg proteins of 250, 150, 100, ~80, and ~65kDa bind to the fusion proteins as fast as 1 minute post sperm addition, while the same 5 bands plus two additional bands at ~200kDa and ~125kDa are seen in eggs at 2, 3, 5, 10, and 20 minutes post sperm addition, with varying intensities. Furthermore, tyrosine phosphorylated proteins from ~70kDa to ~45kDa are seen to bind with varying intensity at 5, 10, and 20 minutes post sperm addition. These findings indicate that multiple proteins are interacting with the SFK Fyn in a fertilization‐specific manner. In future experiments, mass spectrometry and mRNA knock down strategies will be used to identify these proteins and to discover their function in regulating Ca2+ release at fertilization.Support or Funding InformationThis work was funded by the NSF and Florida Institute of Technology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.