Low density detergent-insoluble membrane of Xenopus eggs: subcellular microdomain for tyrosine kinase signaling in fertilization.
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 alpha, Ras, integrin beta 1 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-beta-cyclodextrin, a cholesterol-binding substance, led to a decrease in cholesterol, Xyk and sperm-induced tyrosine phosphorylation in LD-DIM. In methyl-beta-cyclodextrin-treated eggs, sperm-induced Ca(2+) 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.
Sato
Ken-ichi
K
Research Center for Environmental Genomics, Kobe University, Nada, Kobe 657-8501 Japan. kksato@kobe-u.ac.jp
Iwasaki
Tetsushi
T
Ogawa
Keiko
K
Konishi
Masako
M
Tokmakov
Alexander A
AA
Fukami
Yasuo
Y
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002499:Centrifugation, Density Gradient / Q000379:methods; D003505:Cyclodextrins / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D005260:Female; D005306:Fertilization; D008297:Male; D021962:Membrane Microdomains / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology; D010063:Ovum / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D012995:Solubility; D013084:Sperm-Ovum Interactions / Q000502:physiology*; D013094:Spermatozoa / Q000378:metabolism / Q000502:physiology; D013347:Subcellular Fractions; D014982:Xenopus laevis; D047392:beta-Cyclodextrins; D019061:src-Family Kinases
D003505:Cyclodextrins; D003902:Detergents; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D017830:Octoxynol; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D002118:Calcium