Reconstitution of Src-dependent phospholipase Cγ phosphorylation and transient calcium release by using membrane rafts and cell-free extracts from Xenopus eggs

Abstract

We reported previously that egg membrane rafts serve as a subcellular microdomain for sperm-dependent tyrosine kinase signaling in Xenopus fertilization. Moreover, we demonstrated that raft-associated Src tyrosine kinase was activated by sperm in vitro. Here we show that egg rafts incubated with sperm or hydrogen peroxide (H2O2) can promote Src-dependent phosphorylation of phospholipase Cγ (PLCγ) and transient calcium release in the extracts of unfertilized Xenopus eggs. In vivo egg activation by sperm or H2O2 also promotes tyrosinephosphorylation and raft-translocalization of PLCγ. Immunodepletion of PLCγ from the egg extracts inhibits the raft-dependent calcium release. Rafts prepared from H2O2-activated eggs also promote Src-dependent dephosphorylation of p42 mitogen-activated protein kinase and cell cycle transition from metaphase II to interphase in egg extracts. PLCγ phosphorylation and calcium release in egg extracts can be promoted by rafts prepared from COS-7 cells expressing the Xenopus Src gene. These results demonstrate that the signaling events elicited by fertilization in Xenopus eggs can be reconstituted in vitro. The development of such experimental platforms will allow us to dissect the molecular mechanism of sperm-dependent activation of raft-associated Src and subsequent up-regulation of PLCγ and egg activation machinery in Xenopus eggs.