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J. Biol. Chem., Vol. 278, Issue 40, 38413-38420, October 3, 2003

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Reconstitution of Src-dependent Phospholipase C Phosphorylation and Transient Calcium Release by Using Membrane Rafts and Cell-free Extracts from Xenopus Eggs^*

Ken-ichi Sato   ¶, Alexander A. Tokmakov ||, Chang-Li He **, Manabu Kurokawa **, Tetsushi Iwasaki , Mikako Shirouzu ||, Rafael A. Fissore **, Shigeyuki Yokoyama || and Yasuo Fukami  

From the Research Center for Environmental Genomics, Department of Biology, Faculty of Science, Kobe University, Nada, Kobe 657-8501, Japan, the ^||Genome Sciences Center, RIKEN Yokohama Institute, Tsurumi, Yokohama, 230-0045, Japan, and the ^**Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003

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 C (PLC) 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 PLC. Immunodepletion of PLC 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. PLC 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 PLC and egg activation machinery in Xenopus eggs.

Received for publication, March 14, 2003 , and in revised form, June 30, 2003.

^* This work was supported in part by Priority Area-A "Developmental Dynamics" Grant 13045026 and Promotion Research for Young Scientists Grant 12780556 from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and a grant for Scientist Exchange in Japan (NCI-JSPS) Cooperative Cancer Research Program (to K. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 81-78-803-5953; Fax: 81-78-803-5951; E-mail: kksato{at}kobe-u.ac.jp.

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