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Originally published In Press as doi:10.1074/jbc.M302617200 on July 7, 2003

J. Biol. Chem., Vol. 278, Issue 40, 38413-38420, October 3, 2003
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Reconstitution of Src-dependent Phospholipase C{gamma} Phosphorylation and Transient Calcium Release by Using Membrane Rafts and Cell-free Extracts from Xenopus Eggs*

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

From the {ddagger}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

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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{gamma} (PLC{gamma}) 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{gamma}. Immunodepletion of PLC{gamma} 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{gamma} 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{gamma} and egg activation machinery in Xenopus eggs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Fertilization is triggered by the interaction of gametes followed by a subsequent increase in the free calcium concentration ([Ca2+]i)1 within the egg cytoplasm. This paradigm of initiation of development is conserved evolutionarily in a wide range of higher eukaryotic species, from worms to mammals and plants. Whereas the molecular diversity of the gameteinteracting machinery guarantees a species-specific gamete interaction, there seems to exist a canonical series of intracellular events accompanying the fertilization-induced Ca2+ signaling process. Namely, fertilized eggs undergo a transient Ca2+ release at least in part through the inositol 1,4,5-trisphosphate (InsPtd(1,4,5)P3)-dependent pathway (1, 2). This Ca2+ release signal is necessary and sufficient to initiate a series of events associated with embryonic development, such as entry into the somatic cell cycle that involves the completion of the meiotic cell cycle and de novo DNA synthesis (3, 4).

Several lines of evidence indicate that a certain kind(s) of phospholipase C (PLC), a family of enzymes that produce InsPtd(1,4,5)P3 and diacylglycerol from phosphatidylinositol 4,5-bisphosphate, may play a direct role in the sperm-induced production of InsPtd(1,4,5)P3 in the eggs of sea urchin (5–8), starfish (9, 10), ascidian (11), frog (12), and mouse (13–15). PLCs now comprise at least five subspecies: {beta}, {gamma}, {delta}, {epsilon}, and {zeta}. However, the precise roles of each PLC subspecies, the mechanisms of activation, and the source of the relevant PLCs (from sperm, eggs, or both) in sperm-induced InsPtd(1,4,5)P3 production and Ca2+ release remain controversial (16, 17).

Fertilization is accompanied by a rapid and transient increase of tyrosine-phosphorylated proteins in the eggs, indicating that protein-tyrosine kinases (PTKs) are activated upon fertilization. Fertilization-induced protein-tyrosine phosphorylation is synchronous with, or even earlier than, the release of Ca2+, suggesting that tyrosine phosphorylation is upstream of Ca2+ release. PTK inhibitors or recombinant proteins that antagonize enodogenous PTK function have been shown to block sperm-induced Ca2+ release in sea urchin (18, 19), starfish (20), ascidian (21), frog (12, 22–24), and mouse (13)2 eggs. Ectopic expression or microinjection of the active forms of PTKs (10, 25, 26) and artificial up-regulation of PTKs of the egg (27, 28) have been shown to cause parthenogenetic egg activation accompanied by Ca2+ release. Thus, activation of PTKs in eggs may be necessary and sufficient to cause Ca2+ release, although the molecular identities of the responsible egg PTKs have not been precisely defined.

In eggs of the African clawed frog, Xenopus laevis, a 57-kDa Src tyrosine kinase (29),3 which we have formerly denoted Xyk, is activated within 1 min of fertilization (30), and the {gamma}-subspecies of PLC (PLC{gamma}) becomes tyrosine-phosphorylated, activated, and interact with Src within 2 min of fertilization (12). Inhibition of the Src activity of the egg blocks tyrosine phosphorylation and activation of PLC{gamma}, production of InsPtd(1,4,5)P3, Ca2+ release, and resumption of the meiotic cell cycle (12, 24), suggesting that the Src-PLC{gamma} signaling cascade is involved in fertilization.

Xenopus egg Src localizes primarily to egg cortical membranes, and a portion of the fertilization-activated Src undergoes a translocation from the membrane to the cytosolic fraction (24, 30). More recently, we have found that this PTK is enriched in low-density detergent-insoluble membrane fractions of eggs (31). The Xenopus eggs low density detergent-insoluble membrane fractions are also enriched in cholesterol and the ganglioside GM1, indicating that they have characteristics of membrane "rafts" (32, 33). Rafts appear to have a primary role in Xenopus egg fertilization, because sperm-induced protein-tyrosine phosphorylation, including that of Src, takes place in rafts (31). In addition, the cholesterol-binding compound methyl-{beta}-cyclodextrin abolishes sperm-egg interaction and/or subsequent Src activation (31). The functional importance of egg rafts has also been suggested in sea urchin fertilization (34). However, the significance of the structural and functional interaction of rafts with the egg cytoplasm and the involvement of Src-PLC{gamma} pathway in this interaction remain to be established.

For the convenience of fertilization signaling analysis, it would certainly be helpful to develop a cell-free system in which signaling events of fertilization can be analyzed. Egg extracts from unfertilized Xenopus eggs, called CSF (cytostatic factor-arrested) extracts, are known to undergo the Ca2+-dependent cell cycle transition as seen in fertilized eggs (35–37). On the other hand, our previous study has demonstrated that sperm-egg interaction and Src signaling can be reconstituted using isolated rafts (31). We therefore made an effort to investigate whether isolated rafts and CSF extracts can reconstitute the functional interaction between Src and PLC{gamma} leading to a transient Ca2+ release and cell cycle transition. Experiments designed to determine the role of Src and PLC activity revealed a novel role of rafts and a functional cross-talk between rafts and egg extracts, which may operate in physiological Xenopus egg fertilization.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Antibodies—X. laevis were purchased from a domestic dealer (Hamamatsu Seibutsu Kyozai, Hamamatsu, Japan). Anti-Src tyrosine kinase polyclonal antibody, anti-pepY antibody, which has been shown to recognize Xenopus Src (24), was raised against a synthetic peptide that corresponds to residues 410–428 of the chicken c-Src according to the described method (38). A monoclonal antibody against mammalian PLC{gamma} was purchased from Upstate Biotechnology (Lake Placid, NY). Monoclonal anti-phosphotyrosine antibody PY99 was from Santa Cruz Biotechnology (Santa Cruz, CA). An antibody against phosphorylated MAP (mitogen-activated protein) kinase was from New England Biolabs (Beverly, MA). Anti-FLAG antibody (M2 clone) was from Sigma.

Chemicals—Leupeptin was purchased from Peptide Institute (Osaka, Japan). 4-Amidinobenzylsulfonyl fluoride hydrochloride (APMSF) was obtained from Wako Pure Chemicals (Osaka, Japan). H2O2 was purchased from Santoku Chemical Industries (Tokyo, Japan). The Ca2+ ionophore A23187 [GenBank] and a synthetic Arg-Gly-Asp-Ser (RGDS) peptide were from Sigma. cAMP and GTP{gamma}S were from Calbiochem (San Diego, CA). [{gamma}-32P]ATP (>7,000 Ci/mmol) was purchased from ICN Biomedicals (Costa Mesa, CA). A tyrosine kinase substrate peptide, Cdc2 peptide, and Src inhibitor peptide, peptide A7, and its inactive derivative, peptide A9, were prepared as described previously (39). PP2, PP3, U-73122, U-73343, and heparin were from Calbiochem. Fura 2 was from Molecular Probes (Eugene, OR). Other chemicals were from Nacalai Tesque (Kyoto, Japan), Sigma, or Wako.

Eggs, Egg Activation, and Sperm—Unless otherwise stated, all procedures were carried out at ambient temperature. To obtain eggs, Xenopus adult females were injected subcutaneously with 40–80 units of pregnant mare serum gonadotropin. After 3–6 days of the injection, the same animals were induced to ovulate with an injection of 500 units of human chorionic gonadotropin (Teikokuzoki, Tokyo, Japan). Ovulation began 6–8 h after the second injection. Eggs were washed three times with DeBoer's buffer (DB: 110 mM NaCl, 1.3 mM KCl, 0.44 mM CaCl2, pH 7.2–7.4, adjusted by NaHCO3) and then gently incubated with more than a 2-fold volume of DB supplemented with 2% cysteine and 0.06 N NaOH, pH 7.8, for 3–5 min. The resulting dejellied eggs were used within 2 h. Eggs were activated by jelly water-treated Xenopus sperm (see below), H2O2, or A23187 [GenBank] , as described previously (31). After the activation treatment, eggs were immediately washed with DB, snap frozen in liquid nitrogen, and kept at –80 °C. Unfertilized eggs in the same batch of the preparation were also frozen, and kept at –80 °C as control. To obtain sperm, a pair of testes were removed from Xenopus adult males, minced, and washed twice with ice-cold DB. The washed sperm (about 108 sperm from one animal) was incubated with excess volume of Xenopus egg jelly water for 10 min. After the incubation, the mixture was centrifuged at 2,000 x g for 10 min. The pellet was resuspended with 1 ml of DB and was designated jelly water-treated sperm. Demembranated sperm nuclei were prepared according to the described methods (37). Briefly, the washed sperm pellet was resuspended with 1 ml of nuclear preparation buffer (250 mM sucrose, 15 mM Hepes-NaOH, 1 mM EDTA, 0.5 mM spermidine trihydrochloride, 0.2 mM spermidine tetrahydrochloride, 1 mM dithiothreitol, 3% bovine serum albumin, 10 µg/ml leupeptin, 20 µM APMSF, pH 7.4). The suspension was added with lysolecithin (0.5 mg/ml) and incubated for 5 min. After the incubation, the suspension was added to 10 ml of ice-cold nuclear preparation buffer and centrifuged at 2,000 x g for 10 min at 4 °C. The pellet fraction was resuspended with 1 ml of nuclear preparation buffer containing 30% glycerol, and stored at –80 °C until required.

Egg Low Density Detergent-insoluble Membrane (Rafts)—Egg rafts were prepared as described previously (31) with some modifications. All manipulations were conducted on ice or at 4 °C. Eggs were mixed with 5-fold volume of ice-cold buffer A (20 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 10 mM {beta}-mercaptoethanol, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, 20 µM APMSF, pH 7.5) supplemented with 150 mM NaCl and 250 mM sucrose, and homogenized with a 7-ml Dounce tissue grinder (Wheaton, Millville, NJ). The homogenates were centrifuged at 500 x g for 10 min, and the supernatants were collected and centrifuged at 150,000 x g for 20 min. Concentrated Triton X-100 (25%) was then added to the fluffy layer of the pellet (crude membranes) to yield a final concentration of Triton X-100 at 1%. The mixtures were homogenized again, incubated on ice for 10 min, and mixed with equal volumes of ice-cold buffer A containing 150 mM NaCl and 85% sucrose. The resulting mixtures (5 ml) were layered first with 19 ml of 30% sucrose and second with 12 ml of 5% sucrose in the same buffer. The samples were centrifuged at 144,000 x g for 20–24 h in an SW28 rotor (Beckman, Palo Alto, CA). After the centrifugation, 3-ml aliquots of 12 fractions were collected from the top to the bottom of the tubes. Fractions 3–6 were pooled as egg rafts, whereas fractions 10–12 were pooled as detergent-soluble non-raft fractions. In some experiments, egg rafts were diluted with water more than 4-fold and centrifuged at 150,000 x g for 30 min. Such concentrated rafts were resuspended with 200 µl of buffer A containing 150 mM NaCl, and used for experiments.

In Vitro Src Tyrosine Kinase Assay—Src tyrosine kinase activity in rafts was determined by an in vitro kinase assay using Cdc2 peptide as an exogenous substrate (24). Concentrated raft fractions (10 µl) were preincubated with or without raft activators such as 106/ml sperm, 1 mM GTP{gamma}S,1mM cAMP, 1 mM RGDS peptide, or 1 mM CaCl2 for 10 min at 30 °C. The mixtures were further incubated at 30 °C for 10 min in the presence of 5 mM MgCl2, 20 mM Tris-HCl, 1 mM dithiothreitol, 2 µM [{gamma}-32P]ATP (10 µCi), and 1 mM Cdc2 peptide. The reaction was stopped by the addition of Laemmli's SDS sample buffer (40) followed by heat treatment at 98 °C for 5 min. 32P-Labeled Cdc2 peptide was separated by SDS-PAGE on 16% gels and analyzed by a BAS2000 Bioimaging analyzer (Fuji Film, Tokyo, Japan). A similar experiment was conducted to determine Src tyrosine kinase activity that was released from the rafts. In this case, raft samples (50 µg, 100 µl) were mixed with 1 ml of buffer A containing 150 mM NaCl, and centrifuged at 150,000 x g for 10 min. The resulting supernatants (100 µl) were incubated with 1 µlof anti-pepY antibody for 3 h at 4 °C. The immune complexes were adsorbed onto protein A-Sepharose beads (Amersham Biosciences), washed, and subjected to incubation in the presence of [{gamma}-32P]ATP and Cdc2 peptide as described above.

Tyrosine Phosphorylation of PLC{gamma}—Rafts alone (3 µl), CSF extracts alone (27 µl), or a mixture of both were incubated in the absence or presence of 1.5 µl of the activators (see above) and/or 1.5 µl of various inhibitors: 10 µM PP2, 10 µM PP3, 10 µM U73122 [GenBank] , 10 µM U73343 [GenBank] , 100 µM heparin, or 5 mM EGTA, for 10 min at 30 °C. The mixtures were then incubated with 5 mM MgCl2 and 1 mM ATP for 10 min. The kinase reaction was terminated by the addition of 30 µl of 10 mM EDTA on ice. Proteins were solubilized by incubation in the presence of 0.1% SDS and 1 mM sodium orthovanadate at 37 °C for 10 min, and collected as the supernatant fractions by centrifugation at 150,000 x g for 10 min at 4 °C. The supernatant fractions were subjected to immunoprecipitation with anti-PLC{gamma} antibody followed by immunoblotting to analyze the amount as well as the extent of tyrosine phosphorylation of PLC{gamma}. Alternatively, phosphorylation of PLC{gamma} was determined in rafts and non-rafts that had been prepared from several egg samples. In this case, 10 µg of protein from the SDS-solubilized rafts and 500 µg of proteins from the Triton X-100-soluble non-rafts were subjected to immunoprecipitation and immunoblotting.

Immunoprecipitation, SDS-PAGE, and Immunoblotting—Protein samples (50–500 µg, specified in the text) were immunoprecipitated with 1 µl of anti-pepY antiserum or 3 µg of anti-PLC{gamma} antibody for 3 h at 4 °C. After centrifugation at 10,000 rpm for 10 min at 4 °C, the immune complexes were adsorbed onto 10 µl of protein A-Sepharose beads by gentle agitation for 30 min at 4 °C. The beads were washed three times with 500 µl of RIPA buffer, washed once with buffer A, and used as immunoprecipitates. The immunoprecipitates prepared as above were treated with Laemmli's SDS sample buffer (42) at 98 °C for 5 min. SDS-PAGE and immunoblotting of the SDS-denatured proteins was done as described previously (12, 24). Phosphorylation of p42 MAP kinase was analyzed by direct application of protein samples to SDS-PAGE on 8% gels.

Immunodepletion of PLC{gamma}—Depletion of PLC{gamma} was done by incubating CSF extracts (100 µl) with anti-PLC{gamma} antibody (1 µl) for 3 h at 4 °C followed by the adsorption of the immune complexes onto protein ASepharose beads. The resulting supernatant fractions were used as CSF extracts depleted of PLC{gamma} (CSF/{Delta}PLC{gamma}). The efficiency of the depletion was determined by densitometric scanning of the anti-PLC{gamma} immunoblotting data for intact CSF extracts and CSF/{Delta}PLC{gamma}.

Other Methods—Preparation of CSF extracts, measurement of Ca2+ release, and cell cycle transition assay were carried out as described (41, 42). Cloning of Xenopus Src gene, construction of vectors expressing FLAG-tagged Xenopus Src, wild type and kinase-negative version, in which Lys-294 is replaced by Met, and isolation of rafts from COS7 cells expressing FLAG-tagged Xenopus Src will be described elsewhere.4


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Sperm Activates Src Tyrosine Kinase in Isolated Egg Rafts—We first examined the activity of the raft-associated Src in the presence of sperm and an exogenous Src substrate, Cdc2 peptide. As shown in the left panel of Fig. 1A, peptide phosphorylation was stimulated about 3-fold by the sperm. A similar extent of activation was observed with GTP{gamma}S, a G-protein activator that has been shown to activate Xenopus eggs (see "Discussion"). On the other hand, cAMP, an integrin-interacting peptide RGDS, and CaCl2 did not show such an effect (Fig. 1A). The Src-specific inhibitor PP2 completely blocked the peptide phosphorylation induced by either sperm or GTP{gamma}S (data not shown), suggesting that raft-induced peptide phosphorylation is mediated by Src (see "Discussion").



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  		FIG. 1.
Activation and translocation of Src and phospholipase C{gamma}. A, left panels, rafts (0.5 µg of protein that corresponds to about 5–10 eggs, 10 µl) from unfertilized eggs were preincubated for 10 min with sperm (106/ml), GTP{gamma}S(1mM), cAMP (1 mM), RGDS (1 mM), or CaCl2 (1 mM), and then subjected to in vitro kinase assay with Cdc2 peptide as an exogenous substrate. Right panels, rafts preincubated as in the left panel were diluted and ultracentrifuged. The resulting supernatants were immunoprecipitated with anti-Src antibody, and the immunoprecipitated Src activity was assessed by in vitro kinase assay. Upper panels show representative images for phosphorylated Cdc2 peptide (32P-peptide, indicated by arrowheads). Bars in the lower graphs show mean ± S.D. of three independent experiments. Bars with an asterisk indicate that the data are significantly different from control (p < 0.01). In both experiments, sperm alone did not show any peptide phosphorylation activity. B, rafts from unfertilized eggs (Uf-rafts, 0.5 µg of protein) and/or CSF extracts (500 µg of protein that corresponds to about 4–8 eggs) were preincubated with activators as in panel A and then subjected to in vitro kinase assay. The reaction mixtures were treated with EDTA and SDS, diluted, and subjected to immunoprecipitation (IP) with anti-PLC{gamma}. The immunoprecipitates were analyzed by immunoblotting (IB) with either anti-phosphotyrosine antibody (Tyr(P), PY99 IgG at 0.2 µg/ml), or anti-PLC{gamma} antibody (1 µg/ml IgG). The positions of tyrosine-phosphorylated PLC{gamma} (P-PLC{gamma}) and total PLC{gamma} are indicated. C, mixtures of unfertilized egg rafts (0.5 µg of protein) and CSF extracts (50 µg of protein) were preincubated with sperm (106/ml), and then subjected to in vitro kinase assay in the presence of various compounds: PP2 (10 µM), PP3 (10 µM), U73122 [GenBank] (10 µM), U73343 [GenBank] (10 µM), heparin (100 µM), and EGTA (5 mM). Tyrosine phosphorylation of PLC{gamma} was analyzed as in panel B. D, rafts (50 µg of protein) and non-raft fractions (500 µg of protein) were prepared from Xenopus eggs at different time points after insemination. The extent of tyrosine phosphorylation and total amount of PLC{gamma} in each fraction were assessed by immunoprecipitation and immunoblotting as in panel B.

 

We then determined whether Src is released from rafts in the presence of activators. Mixtures of rafts and activators were ultracentrifuged, supernatant fractions were immunoprecipitated with an anti-Src antibody, and the immunoprecipitates were subjected to an in vitro kinase assay. As shown in the right panel of Fig. 1A, sperm, cAMP, and RGDS, but neither GTP{gamma}S nor CaCl2, caused a release of peptide kinase activity from rafts (see "Discussion"). In these experiments, Src activity was not detectable in sperm alone controls.

Raft-associated Src Promotes Phosphorylation of PLC{gamma} in CSF Extracts—To determine whether the activation of raft-associated Src is relevant to downstream signaling events, we analyzed tyrosine phosphorylation of PLC{gamma} in unfertilized egg extracts (CSF extracts). Equivalent amounts of rafts, CSF extracts, or mixtures of both were subjected to the in vitro kinase assay in the presence of various activators. As shown in Fig. 1B, tyrosine phosphorylation of PLC{gamma} was seen only when rafts and CSF extracts were co-incubated in the presence of either sperm or GTP{gamma}S, whereas cAMP and CaCl2 were ineffective. The sperm-dependent tyrosine phosphorylation of PLC{gamma} could be abolished by PP2, but not by the PLC inhibitor U-73122 or heparin, an antagonist for InsPtd(1,4,5)P3 receptor (Fig. 1C). We also examined the subcellular localization of PLC{gamma} before and after fertilization of Xenopus eggs. As shown in Fig. 1D, tyrosine-phosphorylated PLC{gamma} was transiently accumulated in rafts, suggesting that PLC{gamma} undergoes a translocation upon fertilization.

Raft Localization of Active Src and PLC{gamma} in Activated Eggs—We next analyzed a relationship between Src activation and the localization of PLC{gamma} in activated eggs. As shown in Fig. 2A, rafts from fertilized eggs (F-rafts) and H2O2-activated eggs (H-rafts), but not rafts from A23187 [GenBank] -treated eggs (A-rafts), showed a marked increase in Src activity when compared with those from unfertilized eggs (Uf-rafts). The extent of Src activation in the H-rafts was about 2.5-fold higher than that in the F-rafts (Fig. 2A). The Src activation was accompanied by an increase in autophosphorylation of Src (lower panels). F-rafts and H-rafts were found to contain tyrosine-phosphorylated PLC{gamma}, and H-rafts were more prominent in the phosphorylation and translocation of PLC{gamma} than F-rafts (Fig. 2B).



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  		FIG. 2.
Up-regulation of raft-associated Src and tyrosine phosphorylation of PLC{gamma} in activated Xenopus eggs. A, rafts (0.5 µg of protein) from unfertilized (Uf), fertilized (F, 107/ml sperm, 5 min), A23187 [GenBank] -activated (A, 0.5 µM, 5 min), or H2O2-activated eggs (H, 10 mM, 5 min) were subjected to in vitro kinase assay. 32P incorporation into Cdc2 peptide was quantified by a BAS2000 Bioimaging analyzer. The data represent mean ± S.D. of four independent experiments. *, p < 0.01. The raft preparations were also directly analyzed by immunoblotting with either anti-Src antibody (Src) or anti-Tyr(P) 416 antibody (pY416). B, rafts (5 µg of protein) and non-rafts (500 µg of protein) prepared from egg samples were analyzed for tyrosine phosphorylation of PLC{gamma} as in Fig. 1D.

 

Raft-dependent Ca2+ Release in CSF Extracts via the Src-PLC{gamma}-InsPtd(1,4,5)P3 Pathway—We then examined the ability of rafts to cause Ca2+ release in CSF extracts. CSF extracts can show InsPtd(1,4,5)P3-dependent Ca2+ release that is sensitive to heparin, an antagonist for InsPtd(1,4,5)P3 receptor, but not to PP2 (Fig. 3A). As shown in Fig. 3B, sperm-incubated rafts also induced a significant increase in [Ca2+]i in CSF extracts. The mean change in the Fura 2 ratio signal was 0.08 ± 0.01 (n = 9), which corresponded to an increase in [Ca2+]i of 200–300 nM, as estimated with a calibrated curve (41). PP2 and U-73122, but not their inactive analogues PP3 and U-73343 (not shown), and heparin effectively blocked Ca2+ release by sperm-incubated rafts (Fig. 3B). These results suggest that sperm-stimulated rafts lead to Ca2+ release in CSF extracts through the Src-PLC{gamma}-InsPtd(1,4,5)P3 pathway. Consistently, rafts treated with GTP{gamma}S, an activator for raft-associated Src (see Fig. 1, A and B), also induced Ca2+ release in CSF extracts (Fig. 3C).



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  		FIG. 3.
Raft-dependent Ca2+ release in the egg extracts. A,Ca2+ release in CSF extracts was triggered by application of InsPtd(1,4,5)P3 (10 µM) in the absence or presence of PP2 (5 µM) or heparin (100 µM), and monitored by ratio recording of Fura 2 fluorescent signal. B, rafts from unfertilized eggs (Uf-rafts) were preincubated with or without sperm (106/ml) and various inhibitors: PP2 (10 µM), U73122 [GenBank] (10 µM), or heparin (100 µM), and then added to CSF extracts to monitor Ca2+ release as in panel A. C,Ca2+ release was assessed by using GTP{gamma}S(1mM) as a raft activator in the absence or presence of PP2 (10 µM) or U73122 [GenBank] (10 µM). D, rafts from various egg samples (Uf-, F-, A-, and H-rafts) were analyzed for their ability to induce Ca2+ release in CSF extracts. E, H-rafts were subjected to Ca2+ release assay in the absence or presence of PP2 (10 µM), peptide A7 (15 µM), or U73122 [GenBank] (10 µM). Shown are representative traces of the fluorescent ratio signal of Fura 2 as a function of time.

 

We next tested rafts of activated eggs for the Ca2+ release ability. As shown in Fig. 3D, H-rafts induced a prominent rise in [Ca2+]i with a peak amplitude of 0.20 ± 0.03 ratio units (n = 6) that corresponded to 1–2 µM [Ca2+]i. On the other hand, Uf-rafts, F-rafts, and A-rafts were unable to show such a response (Fig. 3D). PP2, peptide A7, another Src-specific peptide inhibitor of 18 amino acids (39), and U-73122 blocked Ca2+ releasing ability of H-rafts (Fig. 3E), whereas PP3, peptide A9 (a 15-amino acid negative control peptide for peptide A7), and U-73343 did not (not shown).

Raft-dependent Cell Cycle Transition in CSF Extracts via Src-PLC{gamma}-InsPtd(1,4,5)P3 Pathway—We performed cell cycle transition assay using demembranated sperm nuclei. The addition of CaCl2 (Fig. 4A) or InsPtd(1,4,5)P3 (not shown) to CSF extracts induced pronuclear formation of exogenously added sperm nuclei, which is indicative of successful cell cycle transition from metaphase II to interphase. Such cell cycle transition was not prevented by PP2 or U-73122 (Fig. 4, B and C), whereas it was prevented by an excess amount of EGTA (not shown). A similar morphological change of added sperm nuclei was observed with rafts prepared from H-rafts (Fig. 4J). However, the efficiency of the cell cycle transition promoted by H-rafts (about 40%) was much lower than that obtained by CaCl2 (100%). Nevertheless, the H-raft-dependent cell cycle transition was sensitive to PP2, peptide A7, and U-73122 (see Fig. 4, K–O), suggesting the involvement of the Src-PLC{gamma}-InsPtd(1,4,5)P3 pathway in this phenomenon. Other raft preparations such as sperm- or GTP{gamma}S-treated Uf-rafts and F-rafts did not promote cell cycle transition (Fig. 4, D–I, see "Discussion").



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  		FIG. 4.
Raft-dependent morphological change of added sperm nuclei in CSF extracts. Cell cycle transition in CSF extracts was determined by morphological changes of added sperm nuclei. A--C, CSF extracts were treated with CaCl2 (1 mM) in the absence (A) or presence of PP2 (B, 10 µM) or U73122 [GenBank] (C, 15 µM) for 40 min. D–F, CSF extracts were treated with Uf-rafts in the absence (D)or presence of sperm (E, 106/ml) or GTP{gamma}S (F, 1 mM). G–J, CSF extracts were incubated with rafts from different egg samples: unfertilized (G), fertilized (H), A23187 [GenBank] -activated eggs (I), and H2O2-treated eggs (J). K–O, CSF extracts were treated with H-rafts in the presence of the inhibitors indicated. Morphologies of demembranated sperm nuclei were determined by staining with Hoechst 33342 followed by observation with fluorescent microscopy. Rounded or swollen sperm nuclei undergo a successful transition (indicated as interphase), whereas condensed or scattered sperm nuclei do not (indicated as Metaphase II).

 

Raft-dependent Dephosphorylation of p42 MAP Kinase in CSF Extracts—As an alternative way to assess Ca2+-dependent signaling, we analyzed the phosphorylation state of p42 MAP kinase. MAP kinase is highly phosphorylated on threonine and tyrosine residues in metaphase II-arrested, i.e. unfertilized, eggs and is completely dephosphorylated after fertilization or H2O2 treatment in a PP2-sensitive manner (Fig. 5, left panel). We found that H-rafts promoted dephosphorylation of p42 MAP kinase in CSF extracts, whereas F-rafts were less effective (Fig. 5, right panel). PP2 effectively blocked the H-raft-induced dephosphorylation of MAP kinase (Fig. 5, right panel).



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  		FIG. 5.
Dephosphorylation of p42 MAP kinase in eggs and CSF extracts. Left panel, dephosphorylation of p42 MAP kinase was determined by immunoblotting (1 µg/ml anti-pMAPK antibody) of Triton X-100-soluble fractions (10 µg of protein) from different egg samples: unfertilized (Uf), fertilized (F), and H2O2-treated eggs (H) that had been premicroinjected with none or PP2 (10 µM). Right panel, dephosphorylation of p42 MAP kinase was determined by immunoblotting of CSF extracts that had been treated with rafts from different egg samples (Uf, F, and H) in the absence or presence of 10 µM PP2 for 40 min. Data shown are representative results of three independent experiments.

 

PLC{gamma} Is Required for Raft-mediated Ca2+ Release in CSF Extracts—To ascertain further the role of PLC{gamma} in the reconstitution system, we proceeded to deplete CSF extracts of PLC{gamma}. Fig. 6A shows that PLC{gamma} could be effectively removed from CSF extracts by using an anti-PLC{gamma} antibody. Immunodepletion with an unrelated antibody did not remove PLC{gamma} from CSF extracts nor did detectable amounts of PLC{gamma} adsorb onto the Sepharose beads (Fig. 6A, Mock). As shown in Fig. 6B, CSF extracts immunodepleted of PLC{gamma}, designated CSF/{Delta}PLC{gamma}, showed an InsPtd(1,4,5)P3-dependent Ca2+ release with a similar kinetics and an amplitude to that observed with intact CSF extracts. However, Ca2+ release induced by sperm-stimulated Uf-rafts was apparently inhibited in CSF/{Delta}PLC{gamma}. On the other hand, H-rafts, which contained tyrosine-phosphorylated PLC{gamma} (see Fig. 2B), induced Ca2+ release in CSF/{Delta}PLC{gamma} as it did in intact CSF extracts (Fig. 6B). Both PP2 and U-73122 could inhibit the H-raft-induced Ca2+ release in CSF/{Delta}PLC{gamma} (not shown).



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  		FIG. 6.
Requirement of PLC{gamma} for raft-dependent Ca2+ release. A, CSF extracts (500 µg of protein) were depleted of endogenous PLC{gamma} by immunoprecipitation (IP) with anti-PLC{gamma} antibody (1 µg of IgG); or mock depleted by immunoprecipitation with control mouse IgG (1 µg of protein) (Mock), as described under "Experimental Procedures." Proteins in the depleted extracts (Sup) and the immune complexes (Ppt) were separated by SDS-PAGE and analyzed by immunoblotting with anti-PLC{gamma} antibody. B, CSF extracts depleted of PLC{gamma} (CSF/{Delta}PLC{gamma}) were subjected to Ca2+ release assay in the absence or presence of InsPtd(1,4,5)P3 (10 µM), Uf-rafts plus either sperm or H-rafts. Control data with the use of intact CSF extracts (gray lines) were also shown.

 

Rafts from COS7 Cells Expressing Src Are Capable of Inducing Ca2+ Release in CSF Extracts—We prepared COS7 cells expressing wild type Xenopus Src. Overexpressed Xenopus Src was found to concentrate to the raft fractions (not shown). Rafts prepared from Src-expressing cells were capable of inducing a significant Ca2+ release in CSF extracts in a PP2-sensitive manner, whereas rafts obtained from control cells were not (Fig. 7A). Pleiotropic effects of PP2 toward PTKs other than Src has recently been inferred (43–45). Therefore we examined the effect of expression of the kinase-negative version of Xenopus Src on Ca2+ release. As we expected, rafts expressing kinasenegative Src did not promote Ca2+ release in CSF extracts (Fig. 7A). Rafts from cells expressing wild type Src, but not kinasenegative Src, induced tyrosine phosphorylation of PLC{gamma} in CSF extracts (Fig. 7B, not shown). PP2 and peptide A7, but not U-73122, effectively blocked the PLC{gamma} phosphorylation (Fig. 7B).



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  		FIG. 7.
Src-dependent Ca2+ release by rafts prepared from COS7 cells. A, rafts prepared from COS7 cells expressing either FLAG-tagged wild type (COS/Src) or kinase-negative (COS/Src-KN) Xenopus Src or vector alone (COS) were subjected to Ca2+ release assay in CSF extracts. Shown are representative traces of the Fura 2 fluorescent ratio signal as a function of time. Effect of PP2 (10 µM) on wild type Src was also examined (COS/Src + PP2). B, mixtures of CSF extracts and COS7 cell rafts were subjected to in vitro kinase assay in the absence or presence of inhibitors as indicated (each at 10 µM). The reaction mixtures were immunoprecipitated with anti-PLC{gamma} antibody and the immunoprecipitates were analyzed for tyrosine phosphorylation of PLC{gamma}. Expression of FLAG-tagged Xenopus Src (Src-FLAG) was also analyzed by immunoblotting the kinase reaction mixtures with anti-FLAG antibody (1 µg/ml IgG).

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study was designed to determine whether rafts isolated from Xenopus eggs are able to reconstitute the series of signaling events associated with fertilization. The in vitro system used here establishes a novel function of rafts in association with unfertilized egg extracts. Namely, sperm-dependent activation of raft-associated Src and subsequent activation of egg extract-derived PLC{gamma} will work for transient calcium release, MAP kinase dephosphorylation, and cell cycle transition. Such reconstitution or replication of egg fertilization in vitro would be important for our understanding of the molecular nature of physiological egg fertilization.

The fact that raft-associated Src is activated by addition of sperm (Fig. 1A) suggests that rafts contain sperm-interacting molecules that regulate Src activity. Membrane receptor-mediated regulation of Src is well established in several cellular functions (46, 47). Xenopus and mouse eggs express several isoforms of the integrin proteins, which have been suggested to mediate interaction with sperm (48, 49). Nonetheless, despite extensive investigation, the role of integrins in egg activation processes, e.g. including the activation of Src, has not been resolved. An alternative mechanism of Src activation in fertilization has been suggested in the mouse (50). In that report, Sette et al. (50) demonstrated that a truncated version of c-Kit tyrosine kinase, which is present in the sperm and would be delivered into the egg after sperm-egg fusion, is capable of activating Fyn, a Src family protein, leading to egg activation and initiation of development. In addition, PLC{zeta}, a novel and sperm-specific type of PLC, has been identified as a sperm-specific egg-activating component in mammals (15). It should be noted that in the case of PLC{zeta}, the involvement of eggassociated Src and other PTKs is uncertain because PLC{zeta} can produce InsPtd(1,4,5)P3 directly. Altogether, whether or not these soluble sperm components are working in Xenopus has not been determined. Thus, two vital questions concerning the mechanism of sperm-induced Src activation that still remain to be answered are as follows. First, what is the molecular mechanism of sperm-egg interaction accompanied by Src activation: sperm-egg receptor interaction, sperm-derived components, or both, and second, how is Src controlled by the upstream components?

Egg GTP-binding protein(s) has been implicated in sperm-induced egg activation in Xenopus and other species (reviewed in Ref. 17). Our present data with the use of GTP{gamma}S suggest that rafts contain GTP-binding proteins that can activate Src. The mechanism of Src activation by GTP{gamma}S is presently unknown. However, it should be noted that Src can be directly activated by the {alpha}-subunit of Gs or Gi in a GTP-dependent manner (51). We are presently analyzing GTP-binding proteins present in rafts and their role in sperm-induced Src activation and Ca2+ signaling.

Sperm, RGDS, and cAMP, but not GTP{gamma}S, were shown to promote a release of Src from rafts (Fig. 1A). Src is known to undergo translocation from the membrane to the cytosolic fraction in eggs activated by sperm or RGDS (24). PKA-dependent translocation of Src has also been demonstrated in the cultured cell system (52). So, it is possible that raft-associated integrins and/or PKA operate in sperm-dependent Src release from rafts. The fact that GTP{gamma}S did not cause the release of Src indicates that Src activation is not sufficient for the release process. Src in rafts was not activated by RGDS, although RGDS has been shown to activate egg Src (24). It may be possible that RGDS would interact with non-raft integrins to activate Src.

Fertilization is accompanied by a transient localization of tyrosine-phosphorylated PLC{gamma} (Figs. 1D and 2B). The results suggest that egg PLC{gamma} is recruited to the rafts, tyrosine-phosphorylated, and activated by Src after fertilization. Consistently, PLC{gamma} in CSF extracts was tyrosine-phosphorylated in a raft-dependent and PP2-sensitive manner (Fig. 1, B and C). Src-dependent activation of PLC{gamma} has been implicated in sperm-induced Ca2+ release in echinoderm and vertebrate eggs (6, 8–10, 12, 17), although the mechanism by which Src activates PLC{gamma} seems to be species-specific. In echinoderm eggs, microinjection of a recombinant protein encoding the PLC{gamma} Src homology 2 (SH2) domain completely abolishes the sperm-induced Ca2+ release. On the other hand, Xenopus eggs and mouse eggs injected with the recombinant PLC{gamma} SH2 are still able to undergo Ca2+ release in response to fertilization (21, 53). Because multiple mechanisms other than the SH2-dependent one can regulate the activity of PLC{gamma} (54), further study should be directed to clarify how PLC{gamma} undergoes translocation and interacts with Src with the use of the reconstitution system.

Rafts prepared from unfertilized eggs and incubated with sperm or GTP{gamma}S induced a transient increase in [Ca2+]i in CSF extracts. The Ca2+ release ability of these raft preparations was effectively suppressed in the presence of PP2, peptide A7, U-73122, or heparin (Fig. 3). These results suggest that rafts are capable of inducing Ca2+ signaling in CSF extracts in a Src-PLC{gamma}-InsPtd(1,4,5)P3-dependent manner. Because neither rafts alone nor CSF extracts alone could induce Ca2+ release in response to sperm or GTP{gamma}S, it can be concluded that functional interaction between the two fractions make it possible to reconstitute the Ca2+ release machinery. This is consistent with the findings that PLC{gamma} is phosphorylated only when both rafts and CSF extracts are present. The inability of CSF extracts immunodepleted of PLC{gamma} to cause Ca2+ release in response to sperm and rafts (Fig. 6) underscores the requirement of PLC{gamma} for raft-dependent Ca2+ release. Src protein expressed in rafts of COS7 cells triggered tyrosine phosphorylation of PLC{gamma} and Ca2+ release in CSF extracts (Fig. 7). The inability of kinase-negative Src to promote Ca2+ release is also important, because this result establishes more specifically the importance of Src kinase activity than the results obtained with PP2, which may inhibit other tyrosine kinases than Src.

Earlier reports (43–45) have pointed to the fact that PP2 and its structurally related, inhibitory analogue PP1 can inhibit some tyrosine kinases other than Src family kinases. They include p38 kinase, casein kinase I, and C-terminal Src kinase (43), c-Abl and c-Kit (44), and the receptor/kinase for platelet-derived growth factor (45). Therefore, the results obtained with PP2 in this study could be because of the inhibition of these kinases that may be present in Xenopus eggs or in the reconstitution systems, although their protein expression and functional properties during egg fertilization have not yet been demonstrated. Further study is certainly needed to clarify whether the PP2-sensitive kinases other than Src are present and up-regulated in fertilized Xenopus eggs and in the reconstitution systems used in this study.

We showed that rafts prepared from H2O2-treated eggs induced a robust increase in [Ca2+]i and cell cycle transition in CSF extracts (Figs. 3, 4, 5). Importantly, these events were sensitive to PP2, peptide A7, U-73122, and heparin, suggesting that the Src-PLC-InsPtd(1,4,5)P3 pathway is required for these events. In fact, H-rafts were highly enriched in active Src proteins and tyrosine-phosphorylated PLC{gamma}. Specifically, the presence of tyrosine-phosphorylated PLC{gamma} may explain why H-rafts could induce Ca2+ release in CSF extracts depleted of PLC{gamma} (Fig. 6). On the other hand, rafts incubated with sperm did not promote cell cycle transition, although it did promote a significant Ca2+ increase. The reason may be explained by the fact that the amplitude of Ca2+ increase by the sperm-stimulated rafts was much lower than those by H-rafts and InsPtd(1,4,5)P3. Such low extent of Ca2+ increase may be because of the low efficiency of Src and PLC{gamma} activation in the sperm-stimulated rafts, and/or because of a loss of important components during the preparation of rafts. We favored the former possibility because H-rafts could induce Ca2+ increase, whose amplitude was sufficient to lead to cell cycle transition. So, the next question will be how the extent of Src and PLC{gamma} activation and the amplitude of Ca2+ increase can be amplified in the sperm-stimulated rafts. In this connection, it would be interesting to analyze the role of InsPtd(1,4,5)P3 (basal level, sensitivity etc.) in the raft-mediated Ca2+ increase in CSF extracts.

In summary, we developed a novel experimental platform for analyzing fertilization signaling, in which isolated rafts and cell-free extracts are combined. Initial characterization of this system established a novel function of rafts: i.e. sperm-dependent Src activation and subsequent signal transduction involving PLC{gamma} activation, Ca2+ release, and cell cycle transition. Further study will therefore aim to clarify the nature of the molecular machinery involved in sperm-egg interaction, Src activation by sperm, Src-PLC{gamma} interaction, and Ca2+-dependent cell cycle regulation in this system. The in vitro reconstitution approach presented here will be useful for dissecting Xenopus fertilization signaling from the time of sperm-egg interaction to the Ca2+-dependent cell cycle transition, and its evolutionary conservation and species-specific features.


   FOOTNOTES
 
* 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. Back

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

1 The abbreviations used are: [Ca2+]i, free calcium concentration; APMSF, 4-amidinobenzylsulfonyl fluoride hydrochloride; CSF, cytostatic factor; DB, DeBoer's solution; InsPtd(1,4,5)P3, inositol 1,4,5-trisphosphate; MAP kinase, mitogen-activated protein kinase; PKA, protein kinase A; PLC, phospholipase C; PTK, protein-tyrosine kinase; RGDS, Arg-Gly-Asp-Ser peptide; F-rafts, rafts from fertilized eggs; H-raft, rafts from H2O2-activated eggs; A-rafts, rafts from A23187 [GenBank] -treated eggs; Uf-rafts, rafts from unfertilized eggs; SH2, Src homology 2; GTP{gamma}S, guanosine 5'-3-O-(thio)triphosphate. Back

2 M. Kurokawa, K. Sato, and R. A. Fissore, unpublished results. Back

3 T. Iwasaki, K. Sato, and Y. Fukami, unpublished data. Back

4 T. Iwasaki, K. Sato, and Y. Fukami, unpublished results. Back


   ACKNOWLEDGMENTS
 
Our cordial thanks to members of the Biosignal Research Center, Kobe University, for their support to use equipment for antibody preparation, cell transfection, and Ca2+ measurements. We also thank Jeremy Smyth for reading the manuscript and for helpful suggestions.



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