Requirement of a Src Family Kinase for Initiating Calcium Release at Fertilization in Starfish Eggs

Andrew F Giusti,David J Carroll,Yama A Abassi,Mark Terasaki,Kathy R Foltz,Laurinda A Jaffe

doi:10.1074/jbc.274.41.29318

Andrew F Giusti, David J Carroll + Show 4 more

Open Access

https://doi.org/10.1074/jbc.274.41.29318

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Abstract

Signal transduction leading to calcium release in echinoderm eggs at fertilization requires phospholipase Cgamma-mediated production of inositol trisphosphate (IP(3)), indicating that a tyrosine kinase is a likely upstream regulator. Because previous work has shown a fertilization-dependent association between the Src homology 2 (SH2) domains of phospholipase Cgamma and a Src family kinase, we examined whether a Src family kinase was required for Ca(2+) release at fertilization. To inhibit the function of kinases in this family, we injected starfish eggs with the SH2 domains of Src and Fyn kinases. This inhibited Ca(2+) release in response to fertilization but not in response to injection of IP(3). We further established the specificity of the inhibition by showing that the SH2 domains of several other tyrosine kinases (Abl, Syk, and ZAP-70), and the SH3 domain of Src, were not inhibitory. Also, a point-mutated Src SH2 domain, which has reduced affinity for phosphotyrosine, was a correspondingly less effective inhibitor of fertilization-induced Ca(2+) release. These results indicate that a Src family kinase, by way of its SH2 domain, links sperm-egg interaction to IP(3)-mediated Ca(2+) release at fertilization in starfish eggs.

Highlights

Signal transduction leading to calcium release in echinoderm eggs at fertilization requires phospholipase C␥mediated production of inositol trisphosphate (IP3), indicating that a tyrosine kinase is a likely upstream regulator
Because previous work has shown a fertilization-dependent association between the Src homology 2 (SH2) domains of phospholipase C␥ and a Src family kinase, we examined whether a Src family kinase was required for Ca2؉ release at fertilization
A point-mutated Src SH2 domain, which has reduced affinity for phosphotyrosine, was a correspondingly less effective inhibitor of fertilization-induced Ca2؉ release. These results indicate that a Src family kinase, by way of its SH2 domain, links sperm-egg interaction to IP3-mediated Ca2؉ release at fertilization in starfish eggs

Summary

EXPERIMENTAL PROCEDURES

GST Fusion Proteins—GST fusion proteins of SH2 domains from various tyrosine kinases were synthesized in bacteria as described previously [7]. DNA encoding the indicated amino acids was obtained as follows. J. Mayer (Harvard Medical School, Boston, MA); human ZAP-70 SH2 (two tandem SH2 domains, amino acids 1–263) was from L. E. Samelson (National Institutes of Health, Bethesda, MD); and rat Syk SH2 (two tandem SH2 domains, amino acids 1–269) was from R. The oocytes were injected with calcium green 10-kDa dextran (Molecular Probes, Eugene, OR). SH2 domain GST fusion proteins were mixed with calcium green dextran, and oocytes were injected with 90 picoliters (3% of their 3100-picoliter volume) to give the indicated cytoplasmic protein concentrations and 10 ␮M calcium green.

RESULTS

DISCUSSION

Src and Fertilization

Calcium green dextran