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Low density detergent-insoluble membrane of Xenopus eggs: subcellular microdomain for tyrosine kinase signaling in fertilization

Ken-ichi Sato^1,2,*, Tetsushi Iwasaki¹, Keiko Ogawa³, Masako Konishi³, Alexander A. Tokmakov^4, and Yasuo Fukami^2,3

¹ Research Center for Environmental Genomics, Kobe University, Nada, Kobe 657-8501 Japan
² Department of Biology, Faculty of Science, Kobe University, Nada, Kobe 657-8501 Japan
³ The Graduate School of Science and Technology, Kobe University, Nada, Kobe 657-8501 Japan
⁴ Biosignal Research Center, Kobe University, Nada, Kobe 657-8501 Japan
Present address: Genomic Sciences Center, RIKEN Yokohama Institute, Yokohama 230-0045 Japan

*Author for correspondence (e-mail: kksato{at}kobe-u.ac.jp)

Accepted 14 November 2001

Protein-tyrosine phosphorylation plays an important role in egg activation signaling at fertilization. We show that in Xenopus, fertilization stimulates a rapid and transient tyrosine phosphorylation of egg proteins, as revealed by immunoblotting with anti-phosphotyrosine antibody. Immunofluorescent microscopic analysis demonstrated that the phosphorylation occurs in cortical area of the egg animal hemisphere. To further characterize subcellular compartment for fertilization-dependent tyrosine kinase signaling, we isolated low density detergent-insoluble membrane (LD-DIM) fraction from Xenopus eggs. The egg LD-DIM was enriched in cholesterol and GM1 ganglioside. It also contained signaling molecules such as Xyk (Xenopus egg Src), Gq, Ras, integrin ß1 and CD9. Fertilization stimulated tyrosine phosphorylation of Xyk and some other LD-DIM proteins. Remarkably, sperm stimulated tyrosine phosphorylation of the LD-DIM proteins in vitro. The sperm-dependent phosphorylation was sensitive to the tyrosine kinase inhibitors PP2 and genistein. We found that pretreatment of eggs with methyl-ß-cyclodextrin, a cholesterol-binding substance, led to a decrease in cholesterol, Xyk and sperm-induced tyrosine phosphorylation in LD-DIM. In methyl-ß-cyclodextrin-treated eggs, sperm-induced Ca²⁺ transient and first cell division were also inhibited. These findings suggest that the egg LD-DIM might serve as subcellular microdomain for tyrosine kinase signaling in Xenopus egg fertilization.

Key words: Fertilization, Egg activation, Protein-tyrosine phosphorylation, Low density detergent-insoluble membrane, Src family kinase, Signal transduction, Xenopus