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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

View larger version (49K): [in a new window]   Fig. 1. Tyrosine phosphorylation of Xenopus egg proteins in response to fertilization, Ca2+ ionophore and H2O2. SDS-solubilized extract was prepared from Xenopus eggs that had been treated with either sperm (insemination, lanes 1-6), A23187 (0.5 µM, lanes 7-12) or H2O2 (10 mM, lanes 13-18) for the indicated times. Egg proteins (each lane contains 50 µg protein that corresponds to one egg) were separated by SDS-PAGE and analyzed by immunoblotting (IB) with either (A) anti-phosphotyrosine antibody (PY99, 1 µg/ml), (B) anti-phosphotyrosine antibody plus 10 mM L-phosphotyrosine or (C) anti-pY416 antibody (10 µg/ml). Asterisks in A indicate the positions of tyrosine-phosphorylated proteins. Black and white arrowheads in A indicate the positions of a 90 kDa protein and a 42 kDa MAP kinase, respectively. Black arrowheads in C indicate the position of Xyk (57 kDa and partially degraded 55 kDa bands). Prestained molecular size markers are maltose binding protein (MBP)-fusion ß-galactosidase (175 kDa), MBP-fusion paramyosin (80 kDa), glutamic dehydrogenase (62 kDa), aldolase (47.5 kDa) and triosephosphate isomerase (32.5 kDa).

View larger version (84K): [in a new window]   Fig. 2. Immunofluorescent microscopic visualization of localized tyrosine phosphorylation in fertilized and H2O2-treated Xenopus eggs. Sections (10 µm) of Xenopus unfertilized eggs (A,B,G,H), fertilized eggs (C-F,I,J; 5 minutes after insemination), A23187-treated eggs (K,L; 0.5 µM, 5 minutes), or H2O2-treated eggs (M,N; 10 mM, 5 minutes) were immunostained with anti-phosphotyrosine antibody (PY99, 1 µg/ml). The samples were analyzed by confocal laser-scanning microscopy (model LSM410, Zeiss, Germany). Indirect immunostained images obtained with fluorescein isothiocyanate (FITC) and the corresponding Nomarski cell images are shown. White arrowheads in C indicate the area showing fertilization-dependent tyrosine phosphorylation. Scale bars: 250 µm in A-F,K-N; 62.5 µm in G-J.

View larger version (61K): [in a new window]   Fig. 3. Isolation and characterization of low density detergent-insoluble membrane (LD-DIM) fraction from Xenopus unfertilized eggs. (A) Schematic diagram for preparation of LD-DIM fraction from Xenopus eggs. (B) Egg LD-DIM fraction, a light-scattering band, is indicated by an asterisk. (C) Protein silver staining of sucrose density gradient fractions. Proteins (20 µl of each fraction) were separated by SDS-PAGE on a 12.5% gel. Prestained molecular size markers are as in Fig. 1 plus ß-lactoglobulin A (25.5 kDa) and lysozyme (16.5 kDa). Each sucrose density gradient fraction was assayed for the content of (D) protein, (E) cholesterol and (F) GM1-ganglioside. (G) Fractions were separated by SDS-PAGE on 8%, 12.5% or 16% gels, and analyzed by immunoblotting with the indicated specific antibodies. An asterisk indicates the position of Xyk in the LD-DIM fractions.

View larger version (35K): [in a new window]   Fig. 4. Fertilization stimulates protein-tyrosine phosphorylation in LD-DIM fractions. Sucrose density gradient fractions (20 µl of each fraction per lane) were prepared from (A) unfertilized, (B) fertilized (5 minutes after insemination) and (C) H2O2-treated (10 mM, 5 minutes) eggs and analyzed by immunoblotting with anti-phosphotyrosine antibody (pTyr, 1 µg/ml of PY99) or anti-pY416 antibody (pY416, 5 µg/ml). Fractions 3-6 of unfertilized and fertilized eggs were concentrated ten times before the experiment. Asterisks indicate the positions of tyrosine-phosphorylated Xyk.

View larger version (46K): [in a new window]   Fig. 5. Sperm stimulates protein-tyrosine phosphorylation in LD-DIM fractions in vitro. Aliquots (50 µl) of LD-DIM fraction (lanes 1-4) or non LD-DIM fraction (lanes 7 and 8) from unfertilized eggs were preincubated in the absence or the presence of jelly water-treated sperm (108 sperm/ml) and subjected to protein kinase assay with or without 5 mM MgCl2 and 1 mM ATP as described in Materials and Methods. As a control, sperm alone was also examined for its kinase activity (lanes 5 and 6). Phosphorylated proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-phosphotyrosine antibody (PY99, 1 µg/ml) without (A) or with (B) 10 mM L-phosphotyrosine. Black asterisks indicate the positions of sperm-dependent phosphorylation bands. A white asterisk indicates the position of a band that can be seen in the presence of sperm alone.

View larger version (48K): [in a new window]   Fig. 6. Characterization of sperm-dependent PTK activity in egg LD-DIM fraction. (A) LD-DIM fraction (50 µl) was preincubated in the absence or the presence of jelly water-treated sperm (108 sperm/ml). The samples were subjected to protein kinase assay in the presence of either DMSO alone (0.2%, lanes 1 and 2), 10 µM PP2 (lane 3), 10 µM PP3 (lane 4), 50 µM genistein (lane 5) or 50 µM daidzein (lane 6). (A) Phosphorylation was carried out with non-labeled ATP and analyzed by anti-phosphotyrosine immunoblotting as in Fig. 5. (B) Phosphorylation was carried out with [-32P]ATP and analyzed by BAS2000 phosphoimaging analyzer. (C) Phosphorylation was performed as in B in the presence of Cdc2 peptide. A phosphoimage for 32P-labeled peptide is shown. (D) LD-DIM fraction was solubilized with extraction buffer containing 2% (v/v) n-octyl-ß-D-glucoside (Wako). The samples (500 µl) were immunoprecipitated with either anti-Xyk antibody (lane 2, 1 µl of rabbit serum) or preimmune antibody (lane 3, 1 µl of rabbit serum). The immunoprecipitates were subjected to protein kinase assay and analyzed by immunoblotting with anti-pY416 antibody (top panel). We also performed protein kinase assay of the immunoprecipitates in the presence of [-32P]ATP and Cdc2 peptide. Autophosphorylated Xyk (middle panel) and phosphorylated peptide (bottom panel) were visualized as in B. Intact LD-DIM fraction was used as a control in each assay (lane 1).

View larger version (39K): [in a new window]   Fig. 7. Methyl-ß-cyclodextrin (MCD) inhibits tyrosine kinase signaling and egg activation in fertilized Xenopus eggs. (A) Xenopus unfertilized eggs were pretreated with 1x DB containing the indicated concentrations of MCD for 60 minutes, washed and inseminated. Appearance of the first cleavage furrow in the eggs was monitored for 100 minutes after insemination. More than 50 eggs were examined in each MCD concentration. (B) LD-DIM fraction was prepared from unfertilized eggs that had been treated with or without MCD as in A. The samples (50 µl each) were subjected to protein kinase assay and analyzed by immunoblotting with anti-phosphotyrosine antibody (PY99, 1 µg/ml). Asterisks indicate the positions of bands that disappear by high dose of MCD. (C) Xenopus unfertilized eggs were pretreated with or without 25 mM MCD for 60 minutes and then left untreated (Uf), or activated by either fertilization (F, 5 minutes of insemination) or H2O2 (H, 10 mM, 5 minutes). LD-DIM fractions (20 µl per lane) were obtained from the egg samples and analyzed for their protein profile by silver staining. (D) LD-DIM fractions (20 µl each) prepared as in C were analyzed by immunoblotting with anti-phosphotyrosine antibody. Asterisks indicate the positions of bands that disappear in the presence of MCD. (E) Eggs were injected with fura-2 and then pretreated with or without 25 mM MCD for 60 minutes. After the pretreatment, eggs were inseminated while recording the fluorescent ratio signal using a high-frame digital CCD imaging ARGUS/HISCA system (Hamamatsu Photonics, Japan). Shown are traces of the fura-2 signal as a function of time. Black arrowheads indicate the time at which insemination was started. In some experiments, A23187 (0.5 µM) was applied after 30 minutes of insemination of MCD-treated eggs (white arrohead). (F) LD-DIM fractions were prepared from untreated and MCD (25 mM)-treated unfertilized eggs and analyzed by immunoblotting with either anti-Xyk (100x dilution of antiserum), anti-integrin ß1(100x dilution of antiserum), anti-Ras (0.5 µg/ml) or anti-Gq (2 µg/ml) antibody. (G) LD-DIM fractions of untreated and MCD-treated eggs prepared as in F were assayed for their relative cholesterol content. The cholesterol content in the untreated egg LD-DIM was taken as 100%. Data represent mean±standard deviations of three independent trials. A bar with an asterisk is significantly different from the control. P <0.01.

View larger version (22K): [in a new window]   Fig. 8. Effect of MCD-mediated removal of cholesterol and its repletion on Xenopus egg fertilization. A group of Xenopus unfertilized eggs (30 eggs) were untreated (control), pretreated with 25 mM MCD (middle) or pretreated with 25 mM MCD then treated with cholesterol (right), and subjected to insemination as in Fig. 7A. The appearance of sperm entry point and cortical contraction (a marker of egg activation) were scored after microscopic observation. Number of positive eggs/total number of eggs tested is indicated.

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