Sato K et al. (2003), Reconstitution of Src-dependent phospholipase C...

XB-ART-5030

J Biol Chem 2003 Oct 03;27840:38413-20. doi: 10.1074/jbc.M302617200.

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Reconstitution of Src-dependent phospholipase Cgamma phosphorylation and transient calcium release by using membrane rafts and cell-free extracts from Xenopus eggs.

Sato K , Tokmakov AA , He CL , Kurokawa M , Iwasaki T , Shirouzu M , Fissore RA , Yokoyama S , Fukami Y .

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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.

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Species referenced: Xenopus
Genes referenced: cdk1 cdk20 src

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	FIG. 1. Activation and translocation of Src and phospholipase Cγ. 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), GTPγS(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-PLCγ. The immunoprecipitates were analyzed by immunoblotting (IB) with either anti-phosphotyrosine antibody (Tyr(P), PY99 IgG at 0.2 μg/ml), or anti-PLCγ antibody (1 μg/ml IgG). The positions of tyrosine-phosphorylated PLCγ (P-PLCγ) and total PLCγ 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 (10 μM), U73343 (10 μM), heparin (100 μM), and EGTA (5 mM). Tyrosine phosphorylation of PLCγ 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 PLCγ in each fraction were assessed by immunoprecipitation and immunoblotting as in panel B.
	FIG. 2. Up-regulation of raft-associated Src and tyrosine phosphorylation of PLCγ in activated Xenopus eggs. A, rafts (0.5 μg of protein) from unfertilized (Uf), fertilized (F, 107/ml sperm, 5 min), A23187-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 PLCγ as in Fig. 1D.
	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 (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 GTPγS(1mM) as a raft activator in the absence or presence of PP2 (10 μM) or U73122 (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 (10 μM). Shown are representative traces of the fluorescent ratio signal of Fura 2 as a function of time.
	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 (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 GTPγS (F, 1 mM). G–J, CSF extracts were incubated with rafts from different egg samples: unfertilized (G), fertilized (H), A23187-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).
	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.
	Requirement of PLCγ for raft-dependent Ca2+ release. A, CSF extracts (500 μg of protein) were depleted of endogenous PLCγ by immunoprecipitation (IP) with anti-PLCγ 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-PLCγ antibody. B, CSF extracts depleted of PLCγ (CSF/δPLCγ) 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.
	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-PLCγ antibody and the immunoprecipitates were analyzed for tyrosine phosphorylation of PLCγ. 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).