Reconstitution of Src-dependent phospholipase Cgamma phosphorylation and transient calcium release by using membrane rafts and cell-free extracts from Xenopus eggs.
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 Cgamma (PLCgamma) and transient calcium release in the extracts of unfertilized Xenopus eggs. In vivo egg activation by sperm or H2O2 also promotes tyrosine phosphorylation and raft-translocalization of PLCgamma. Immunodepletion of PLCgamma 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. PLCgamma 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 PLCgamma and egg activation machinery in Xenopus eggs.
Sato
Ken-ichi
K
Research Center for Environmental Genomics, Faculty of Science, Kobe University, Nada, Kobe 657-8501, Japan. kksato@kobe-u.ac.jp
Tokmakov
Alexander A
AA
He
Chang-Li
CL
Kurokawa
Manabu
M
Iwasaki
Tetsushi
T
Shirouzu
Mikako
M
Fissore
Rafael A
RA
Yokoyama
Shigeyuki
S
Fukami
Yasuo
Y
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002453:Cell Cycle; D002474:Cell-Free System; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D066298:In Vitro Techniques; D008297:Male; D021962:Membrane Microdomains / Q000378:metabolism*; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011233:Precipitin Tests; D021381:Protein Transport; D015398:Signal Transduction; D013094:Spermatozoa; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D015854:Up-Regulation; D014981:Xenopus; D019061:src-Family Kinases / Q000378:metabolism*
D003902:Detergents; D014443:Tyrosine; D006861:Hydrogen Peroxide; D019061:src-Family Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium