Src kinase induces calcium release in Xenopus egg extracts via PLCγ and IP 3-dependent mechanism

Abstract

Mobilization of intracellular calcium is an indispensable step of fertilization-induced egg activation. Recently, this process has been shown to require the sequential activation of Src family tyrosine kinases, phospholipase Cγ (PLCγ), and inositol-1,4,5-trisphosphate (IP 3)-dependent receptor of endoplasmic reticulum. In the present study, we made an attempt to recapitulate the early events of egg activation by stimulating Src kinase activity in the cell-free extracts of Xenopus eggs. We found that enhanced Src kinase activity can initiate calcium response of low magnitude in cytostatic factor (CSF)-arrested mitotic extracts without releasing them into interphase. The addition of catalytically active recombinant Src kinase, as well as the activation of endogenous Xenopus Src family kinase by hydrogen peroxide (H 2O 2), increased total tyrosine phosphorylation, tyrosine phosphorylation of PLCγ, and IP 3 production in the extracts. The treatment with the Src family kinase-specific inhibitor, PP1, or PLC inhibitor, U73122, or IP 3 receptor antagonist, heparin, prevented calcium release in the extracts. We conclude, therefore, that possible mechanism of Src/H 2O 2 action in the extracts might involve tyrosine phosphorylation and activation of PLCγ, accompanied by the increase in IP 3 content and subsequent calcium release from IP 3-regulated calcium stores. These results also suggest that monitoring calcium signals induced in the Xenopus egg extracts by various components of signaling pathways may provide a particularly useful approach to investigating their role in the signal transduction.